Human trk receptors and neurotrophic factor inhibitors

ABSTRACT

The invention concerns human trkB and trkC receptors and their functional derivatives. The invention further concerns immunoadhesins comprising trk receptor sequences fused to immunoglobulin sequences.

This is a continuation of application(s) Ser. No. 08/286,846 filed onAug. 5, 1994 which is a continuation in part of application Ser. No.08/215,139, filed Mar. 18, 1994, now abandoned which applications areincorporated herein by reference and to which application(s) priority isclaimed under 35 USC §120.

FIELD OF THE INVENTION

This invention concerns human trk receptors. The invention furtherconcerns neurotrophic factor inhibitors, and methods for inhibitingneurotrophic factor biological activity.

BACKGROUND OF THE INVENTION

Neurotrophic factors or neurotrophins are a family of small, basicproteins which play a crucial role in the development and maintenance ofthe nervous system. The first identified and probably best understoodmember of this family is nerve growth factor (NGF), which has prominenteffects on developing sensory and sympathetic neurons of the peripheralnervous system (Levi-Montalcini, R. and Angeletti, P.U., Physiol. Rev.48, 534-569 1968!; Thoenen, H. et al., Rev. Physiol. Biochem. Pharmacol.109, 145-178 1987!). Although NGF and a number of animal homologs hadbeen known for a long time, including a homolog from the mousesubmandibular gland, the mature, active form of which is often referredto as β- or 2.5S NGF, it was not until recently that sequentiallyrelated but distinct polypeptides with similar functions wereidentified.

The first in line was a factor called brain-derived neurotrophic factor(BDNF), now also referred to as neurotrophin-2 (NT-2) which was clonedand sequenced by Leibrock, J. et al. (Nature 341, 149-152 1989!). Thisfactor was originally purified from pig brain (Barde, Y. A. et al., EMBOJ. 1, 549-553 1982!), but it was not until its cDNA was cloned andsequenced that its homology with NGF became apparent. The overall aminoacid sequence identity between NGF and BNDF (NT-2) is about 50%. In viewof this finding, Leibrock et al. speculated that there was no reason tothink that EDNF and NGF should be the only members of a family ofneurotrophic factors having in common structural and functionalcharacteristics.

Indeed, further neurotrophic factors closely related to β-NGF and BDNFhave since been discovered. Several groups identified a neurotrophicfactor originally called neuronal factor (NF), and now referred to asneurotrophin-3 (NT-3) (Ernfors et al., Proc. Natl. Acad. Sci. USA 87,5454-5458 (1990); Hohn et al., Nature 344, 339 1990!; Maisonpierre etal., Science 247, 1446 1990!; Rosenthal et al., Neuron 4, 767 1990!;Jones and Reichardt, Proc. Natl. Acad. Sci. USA 87, 8060-8064 (1990);Kaisho et al., FEBS Lett. 187 1990!; copending U.S. application Ser. No.07/494,024 filed Mar. 15, 1990). NT-3 shares about 50% of its aminoacids with both β-NGF and BDNF (NT-2). Neurotrophins-4 and -5 (NT-4 andNT-5), have been recently added to the family (copending U.S.application Ser. No. 07/587,707 filed Sep. 25, 1990; Hallbook, F. etal., Neuron 6, 845-858 19913; Berkmeier, L. R. et al., Neuron 7, 857-8661991! ; Ip et al., Proc. Natl. Acad. Sci USA 89, 3060-3064 1992!). Themammalian molecule initially described by Berkmeier et al. supra, whichwas subsequently seen to be the homolog of Xenopus NT-4, is usuallyreferred to as NT-4/5.

Neurotrophins, similarly to other polypeptide growth factors, affecttheir target cells through interactions with cell surface receptors.According to our current knowledge, two kinds of transmembraneglycoproteins serve as receptors for neurotrophins. Equilibrium bindingstudies have shown that neurotrophin-responsive neurons possess a commonlow molecular weight (65-80 kDa), low affinity receptor (LNGFR), alsotermed as p75^(NTR) or p75, which binds NGF, BDNF, and NT-3 with a K_(D)of 2×10⁻⁹ M, and large molecular weight (130-150 kDa), high affinity(K_(D) in the 10⁻¹¹ M) receptors, which are members of the trk family ofthe receptor tyrosine kinases.

The first member of the trk receptor family, trkA, was initiallyidentified as the result of an oncogenic transformation caused by thetranslocation of tropomyosin sequences onto its catalytic domain. Laterwork identified trka as a signal transducing receptor for NGF.Subsequently, two other related receptors, mouse and rat trkB (Klein etal., EMBO J. 8, 3701-3709 1989!; Middlemas et al., Mol. Cell. Biol. 11,143-153 1991!; EP 455,460 published Nov. 6, 1991) and porcine, mouse andrat trkC (Lamballe et al., Cell, 967-979 1991!; EP 522,530 publishedJan. 13, 1993), were identified as members of the trk receptor family.The structures of the trk receptors are quite similar, but alternatesplicing increases the complexity of the family by giving rise to twoknown forms of trkA, three known forms of trkB (two without functionaltyrosine kinase domains) and at least four forms of trkC (severalwithout functional tyrosine kinase domain, and two with small inserts inthe tyrosine kinase domain). This is summarized in FIG. 1.

The role of the p75 and trk receptors is controversial. It is generallyaccepted that trk receptor tyrosine kinases play an important role inconferring binding specificity to a particular neurotrophin, however,cell lines expressing trka bind not only NGF but also NT-3 and NT-4/5(but not BDNF), trkB expressing cells bind BDNF, NT-3, NT-4, and NT-4/5(but not NGF), in contrast to trkC-expressing cells which have beenreported to bind NT-3 alone (but not the other neurotrophins).Furthermore, it has been shown in model systems that the various formsof trk receptors, arising from alternate splicing events, can activatedifferent intracellular signalling pathways, and therefore presumablymediate different physiological functions in vivo. It is unclear whethercells expressing a given trk receptor in the absence of p75 bindneurotrophins with low or high affinity (Meakin and Shooter, TrendsNeurosci. 15, 323-331 1992!).

Published results of studies using various cell lines are confusing andsuggest that p75 is either essential or dispensable for neurotrophinresponsiveness. Cell lines that express p75 alone bind NGF, BDNF, NT-3,and NT-4 with similar low affinity at equilibrium, but the binding rateconstants are remarkably different. As a result, although p75-binding isa common property of all neurotrophins, it has been suggested the p75receptor may also play a role in ligand discrimination (Rodriguez-Tebaret al., EMBO J. 11, 917-922 1992!). It is unclear whether the p75receptor alone is capable of mediating neurotrophin biological activity.While the trk receptors have been traditionally thought of as thebiologically significant neurotrophic factor receptors, it has recentlybeen demonstrated that in melanoma cells devoid of trkA expression, NGFcan still elicit profound changes in biological behavior presumablythrough p75 (Herrmann et al., Mol. Biol. Cell 4, 1205-1216 1993!).Recently, Davies et al. (Neuron 11, 565-574 1993!) reported the resultsof studies investigating the role of p75 in mediating the survivalresponse of embryonic neurons to neurotrophins in a model of transgenicmice carrying a null mutation in the p75 gene. They found that p75enhances the sensitivity of NGF-dependent cutaneous sensory neurons toNGF.

Neurotrophins exhibit actions on distinct, but overlapping, sets ofperipheral and central neurons. These effects range from playing acrucial role in ensuring the survival of developing neurons (NGF insensory and sympathetic neurons) to relatively subtle effects on themorphology of neurons (NT-3 on purkinje cells). These activities haveled to interest in using neurotrophins as treatments of certainneurodegenerative diseases. Neurotrophins have also been implicated inthe mediation of inflammatory pain, and are overexpressed in certaintypes of malignancies. Accordingly, inhibitors of neurotrophinbiological activity have therapeutic potentials, such as in painmedication and as chemotherapeutics in cancer treatment.

In order to better understand the role of trk and neurotrophin action invarious human pathological states, it would be useful to identify andisolate human trkB and trkC proteins, and specifically, to determinewhich forms of trkB and trkC are expressed in the human. Apart fromtheir scientific and therapeutic potentials, such human trk receptorproteins would be useful in the purification of human neurotrophicfactors, and in the diagnosis of various human pathological conditionsassociated with elevated or reduced levels of neurotrophins capable ofbinding trkB and/or trkC.

It would further be desirable to provide effective inhibitors ofneurotrophic factor biological activity. Such inhibitors would be usefulin the diagnosis and treatment of pathological conditions associatedwith neurotrophic factors.

SUMMARY OF THE INVENTION

The present invention is based on successful research resulting in theidentification, cloning and sequencing of naturally-occurring forms oftrkB and trkC receptors from the human, and in the determination oftheir expression pattern in various tissues by Northern and in situhybridization analysis. The invention is further based onstructure-function mutagenesis studies performed with human trkCreceptor, which resulted in the identification of regions required forreceptor binding and/or biological activity. The invention isadditionally based on the experimental finding that expression of theextracellular domains of human trk receptors as immunoglobulin chimeras(immunoadhesins) leads to soluble molecules which retain the bindingspecificity of the corresponding native receptors and are capable ofblocking the biological activity of their cognate neurotrophins.

In one aspect, the present invention relates to an isolated human trkBor trkC polypeptide selected from the group consisting of:

(a) a native sequence human trkB or trkC polypeptide,

(b) a polypeptide having at least 95% amino acid sequence identity witha native sequence human trkB or trkC polypeptide, exhibiting abiological property of a native human trkB or trkC polypeptide, andbeing non-immunogenic in the human, and

(c) a fragment of a polypeptide of (a) or (b) exhibiting a biologicalproperty of a native human trkB or trkC polypeptide, and beingnon-immunogenic in the human.

In another aspect, the invention concerns antibodies capable of specificbinding any of the foregoing human trkB or trkC polypeptides, and tohybridoma cell lines producing such antibodies.

In yet another aspect, the invention concerns an isolated nucleic acidmolecule comprising a nucleic acid sequence coding for a human trkB ortrkC polypeptide as hereinabove defined.

In a further aspect, the invention concerns an expression vectorcomprising the foregoing nucleic acid molecule operably linked tocontrol sequences recognized by a host cell transformed with the vector.

In a still further aspect, the invention concerns a host celltransformed with the foregoing expression vector.

In a different aspect, the invention concerns a method of using anucleic acid molecule encoding a human trkB or trkC polypeptide ashereinabove defined, comprising expressing such nucleic acid molecule ina cultured host cell transformed with a vector comprising said nucleicacid molecule operably linked to control sequences recognized by thehost cell transformed with the vector, and recovering the encodedpolypeptide from the host cell.

The invention further concerns a method for producing a human trkB ortrkC polypeptide as hereinabove defined, comprising inserting into theDNA of a cell containing nucleic acid encoding said polypeptide atranscription modulatory element in sufficient proximity and orientationto the nucleic acid molecule to influence the transcription thereof.

The invention also provides a method of determining the presence of ahuman trkB or trkC polypeptide, comprising hybridizing DNA encoding suchpolypeptide to a test sample nucleic acid and determining the presenceof human trkB or trkC polypeptide DNA.

In a different aspect, the invention concerns a method of amplifying anucleic acid test sample comprising priming a nucleic acid polymerasereaction with nucleic acid encoding a human trkB or trkC polypeptide, asdefined above.

The invention further concerns an antagonist of a native human trkB ortrkC polypeptide, as hereinabove defined.

In a further embodiment, the invention concerns a pharmaceuticalcomposition comprising (a) a human trkB or trkC polypeptide ashereinabove defined, (b) an antagonist of a native human trkB or trkCpolypeptide, or (c) an antibody specifically binding a polypeptide of(a) or (b), in admixture with a pharmaceutically acceptable carrier.

In yet another aspect, the invention concerns chimeric polypeptidescomprising a trk receptor amino acid sequence capable of binding anative neurotrophic factor, linked to an immunoglobulin sequence. In aspecific embodiment, the chimeric polypeptides are immunoadhesinscomprising a fusion of a trk receptor amino acid sequence capable ofbinding a native neurotrophic factor, to an immunoglobulin sequence. Thetrk receptor is preferably human, and the fusion is preferably with animmunoglobulin constant domain sequence, more preferably with animmunoglobulin heavy chain constant domain sequence. In a particularembodiment, the association of two trk receptor-immunoglobulin heavychain fusions (e.g., via covalent linkage by disulfide bond(s)) resultsin a homodimeric immunoglobulin-like structure. An immunoglobulin lightchain may further be associated with one or both of the trkreceptor-immunoglobulin chimeras in the disulfide-bonded dimer to yielda homotrimeric or homotetrameric structure.

In a further aspect, the invention concerns bispecific moleculescomprising a trk receptor amino acid sequence capable of binding anative neurotrophic factor and a different binding sequence. In aspecial embodiment, such bispecific molecules are immunoadhesinscomprising a fusion of a trk receptor amino acid sequence capable ofbinding a neurotrophic factor to an immunoglobulin sequence covalentlyassociated with a fusion of a different binding sequence to animmunoglobulin sequence. The different binding sequence may, forexample, be a different trk receptor amino acid sequence, capable ofbinding the same or a different neurotrophic factor, or may recognize adeterminant on a cell type expressing the neurotrophic factor to whichthe first trk receptor amino acid sequence binds.

In a preferred embodiment, each of the binding sequences is fused to animmunoglobulin heavy chain constant domain sequence, and the two fusionsare disulfide-bonded to provide a heterodimeric structure.Immunoglobulin light chains may be associated with the bindingsequence-immunoglobulin constant domain fusions in one or both arms ofthe immunoglobulin-like molecule, to provide a disulfide-bondedheterotrimeric or heterotetrameric structure.

The invention further concerns nucleic acid encoding the chimeric chainsof the foregoing mono- or bispecific-immunoadhesins or other bispecificpolypeptides within the scope herein, expression vectors containing DNAencoding such molecules, transformed host cells, and methods for theproduction of the molecules by cultivating transformant host cells.

In a further aspect, the invention concerns a method for purifying aneurotrophic factor by adsorption on an immunoadhesin comprising thefusion of a trk receptor amino acid sequence capable of binding theneurotrophic factor to be purified to an immunoglobulin sequence. Thetrk receptor sequence preferably is of the same species that serves asthe source of the neurotrophic factor to be purified.

In yet another aspect, the invention concerns a method for detecting anucleic acid sequence coding for a polypeptide molecule which comprisesall or part of a human trkB or trkC protein or a related nucleic acidsequence, comprising contacting the nucleic acid sequence with adetectable marker which binds specifically to at least part of thenucleic acid sequence, and detecting the marker so bound.

A method for the diagnosis of a pathological condition characterized bythe over- or underexpression of a neurotrophic factor, comprisingcontacting a biological sample comprising said neurotrophic factor witha detectably labelled trk receptor polypeptide capable of binding saidneurotrophic factor, and detecting the marker so bound.

The invention further concerns pharmaceutical compositions comprising atherapeutically or preventatively effective amount of a mono- orbispecific chimeric polypeptide as hereinabove defined, in admixturewith a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the nucleotide sequence (SEQ ID NO: 1) and deducedamino acid sequence (SEQ ID NO: 2) of human trkB receptor. FIGS. 1A and1B: The sequence of tyrosine kinase domain-containing trkB is shown withpotential N-linked glycosylation sites boxed, predicted transmembranedomain underlined (amino acids 431 to 457 of SEQ ID NO: 2), and tyrosinekinase domain flanked by arrows. The site of the splice giving rise tothe truncated form is indicated by a single vertical line. FIG. 1C: Thesequence (SEQ ID NO: 40) of the alternately spliced truncatedintracellular domain is shown. The amino acid sequence and thenucleotide sequence of the truncated form of human trkB receptor areattached as SEQ. ID. NOS: 4 and 3, respectively.

FIGS. 2A-2C show the nucleotide sequence (SEQ ID NO: 5) amino acid (SEQID NO: 6) sequenc of human trkC receptor. FIGS. 2A and 2B: The sequenceof tyrosine kinase containing trkC is shown with potential N-linkedglycosylation sites boxed, predicted transmembrane domain underlined(amino acids 430 to 453 if SEQ ID NO: 6), and tyrosine kinase domainflanked by arrows. The site of the splice giving rise to the truncatedform is indicated by a single vertical line. The sequence of thepotential inserts in the extracellular and tyrosine kinase domains areflanked by brackets. B) The sequence (SEQ ID NO: 41) of the alternatelyspliced truncated intracellular domain is shown. The amino acid sequenceand the nucleotide sequence of the truncated human trkC receptor areattached as SEQ. ID NOS.: 8 and 7.

FIG. 3. Similarities of various domains of trk family members from ratand human. Percent similarity based on the PAM250 matrix (Dayhoff etal., 1983) was determined for different trk domains as defined bySchneider and Schweiger, Oncogene 6, 1807-1811 (1991). Pairwisecomparison were made between human trkA and human trkB (H A-B), humantrkA and human trkC (H A-C), human trkB and human trkC (H B-C), humantrka and rat trkA (H-R A), human trkB and rat trkB (H-R B), and humantrkC and rat trkC (H-R C).

FIG. 4. Summary of the splice forms seen in human and other mammaliantrks. Shown are schematic representations of the forms of the varioustrks arising from alternate splicing. Domains are after Schneider andSchweiger, supra. Data is redrawn from the literature for rat trkA(Meakin, et al., Proc. Natl. Acad. Sci. USA 89, 2374-2378 1992!, Barkeret al., J. Biol. Chem. 268, 15150-15157 1993!), rat and mouse trkB(Klein, et al., EMBO J. 8, 3701-3709 1989!; Klein et al., Cell 6,647-656 1990!), Middlemas et al., Mol. Cell. Biol. 11, 143-153 1991!)and rat and pig trkC (Lamballe, et al., Cell 66, 967-979 1991!;Valenzuela et al., Neuron 10, 963-974 1993!; Tsoulfas, et al., Neuron10, 975-990 1993!). Alternate forms of truncated rat trkC described byValenzuela et al., supra are omitted for clarity. The closed triangle intrkA extracellular region represents the optionally present peptideSer-Pro-Ser-Arg-Arg-Trp (SEQ ID NO: 39) as described in the text. Thehalf-closed triangle in trkC extracellular region represents theoptionally present 9 amino acid peptide ESTDNFILF (SEQ ID NO: 36) asdescribed in the text. The smaller open triangle in trkC tyrosine kinasedomain represents the optionally present 14 amino acid peptideLFNPSGNDFCIWCE (SEQ ID NO: 37), and the larger open triangle innon-human trkC tyrosine kinase domain represents the optionally present25 or 39 amino acid peptides.

FIG. 5. Amplification of region containing potential insert of tyrosinekinase domain of trkB and trkc. Brain cDNA was amplified with primersselective for the region surrounding the site of the observed insert inthe TK domain of trkC. Using primers selective for trkC, two bands ofsizes corresponding to the no insert (568) or 14 amino acid insert (610)form are amplified, with no evidence for any larger forms. Using primersselective for trkb, only one band corresponding to the no insert form(636) is detected.

FIG. 6. Northern analysis of trk B and trkC expression in human tissues.Two micrograms of poly A+ RNA from the regions indicated was hybridizedwith probes specific for the trkB extracellular domain (ECD) or tyrosinekinase domain (TK) or the trkC extracellular (ECD) or tyrosine kinase(TK) domains. Note that the blot containing the brain regions was imageprocessed differently than those containing the other tissues. In orderto better display the range of hybridization signals present in the widevariety of tissues examined, a higher contrast setting was used for thebrain regions hybridized with the trkB probes and a lower sensitivitywas used for brain regions hybridized with the trkC probes.

FIGS. 7(A-E). In Situ hybridization analysis of embryos and adult brain.In situ hybridization using probes for trkA (FIGS. 7A and 7D)TK-containing trkB (FIG. 7B) and TK-containing trkC (FIGS. 7C and 7E).Shown are sheet film autoradiographs of sagittal sections of eight weekold human embryos (FIGS. 7A, 7B and 7C) with arrowheads pointing todeveloping DRG and asterisks signifying trigeminal ganglion. FIG. 7Dshows hybridization pattern of trkA in a coronal section through nucleusbasalis of Meynert (NBM) and the head of the caudate nucleus (CN), whileFIG. 7E shows the pattern of trkC expression in a coronal sectionthrough hippocampus and adjacent cortex. All scale bars are 500 microns.

FIG. (8A-F). In situ hybridization of developing DRG with trka and trkC.Emulsion autoradiography of developing DRG from human embryos hybridizedwith probes for trka (FIGS. 8A, 8B, and 8C) and trkC (FIG. 8D, 8E, and8F). Ventral is to the right in all panels, and scale bars are 100microns. FIGS. 8A and 8D are darkfield photomicrographs of adjacentsections hybridized with probes for trkA and trk C in rostral DRG. FIGS.8B and 8C and FIGS. 8E and 8F are brightfield and darkfield pairs ofadjacent sections through lumbar DRG hybridized with trkA (FIGS 8B and8C) or trkC (FIGS. 8E and 8F). Note the differential distribution oftrkA and TrkC expressing cells, with trkA expressing cells being moreabundant in the more dorsal aspect of the developing ganglia and trkCexpressing cells more prevalent in the ventral aspect.

FIG. (9A-G). In situ hybridization analysis of expression in areas ofthe adult human nervous system. FIG. 9A shows darkfield photomicrographof hybridization with trkA probe in nucleus basalis of Meynert. FIGS 9Band 9C are a bright and darkfield pair of paraffin section of adult DRGhybridized with TK-containing trkB. Note hybridization only overneurons, and that different neurons show different levels ofhybridization. FIGS. 9D and 9E are bright and dark field pair showinghybridization pattern of TK-containing trkC in parietal cortex. Note themore intense hybridization over layer four and almost complete lack ofhybridization in layer one. FIGS. 9F and 9G are bright and darkfieldpair of trkC in cortex showing hybridization is largely confined tolarge neuron-like cell bodies.

FIG. 10(A-C). Competitive displacement of neurotrophins bound totrk-IgG. Radiolabelled neurotrophins (25 to 35 pM) were bound to trk-IgGin the presence of increasing concentrations of various unlabelledneurotrophins. FIG. 10A: Labelled NGF binding to trkA-IgG. FIG. 10B:Labelled BDNF bound to trkB-IgG. FIG. 10C: Labelled NT3 bound totrkC-IgG. Displacement was with cold NGF (), cold BDNF (∘), cold NT3(▪), or cold NT5 (¤).

FIG. 11(A-C). Neurotrophin bioactivity is blocked by trk immunoadhesins.Neurotrophin bioactivity was assessed by measuring the survival of chickdorsal root (FIGS. 11A and 11B) or sympathetic (FIG. 11C) ganglionneurons in the absence or presence of trk immunoadhesins.

FIG. 12. Structures of trkC deletions and swaps with trkB. Structuraldomains of trkC and trkB in black and grey, respectively.

FIG. 13. Expression of trkC deletions and swaps with trkB. Oneparticular representative experiment is shown. Concentrations weredetermined using an anti-Fc ELISA. Values of trkC variants are expressedas percentage of trkC wild-type expression.

FIG. 14(A-C). Competitive displacement of NT-3 bound to trkC variants.Radiolabeled NT-3 (50pM) was bound to trkC variants in the presence ofincreasing amounts of unlabeled NT-3. (FIG. 14A) Deletions of trkC.(FIG. 11B) Domain swaps of trkC with corresponding sequences from trkB.(FIG. 11C) Variants of Ig-domain 2 of trkC.

FIG. 15(A-C). Competitive displacement of BDNF bound to trkC variants.Radiolabeled BDNF (50pM) was bound to trkC variants in the presence ofincreasing amounts of unlabeled BDNF. (FIG. 15A) Deletions of trkC.(FIG. 15B) Domain swaps of trkC with corresponding sequences from trkB.(FIG. 15C) Swap of Ig-domain 2 with sequence from trkb.

FIG. 16A-16C. Comparison of the amino acid sequences of full lengthhuman trkA, trkB and trkC receptors. The concensus sequences are boxed;the boundaries of the various domains are marked by vertical lines (seeSEQ. ID. NOS: 9, 2 and 6).

FIG. 17. Effect of a trkA-IgG immunoadhesin on carageenan inducedhyperalgesia in rats.

FIG. 18. TrkA-IgG infusion leads to hypoalgesia in rats.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

The terms "neurotrophin" and "neurotrophic factor" and their grammaticalvariants are used interchangeably, and refer to a family of polypeptidescomprising nerve growth factor (NGF) and sequentially related homologs.NGF, brain-derived growth factor (BDNF, a.k.a. NT-2), neurotrophin-3(NT-3), and neurotrophins-4 and -5 (NT-4/5) have so far been identifiedas members of this family.

The terms "neurotrophin" and "neurotrophic factor" include nativeneurotrophins of any (human or non-human) animal species, and theirfunctional derivatives, whether purified from a native source, preparedby methods of recombinant DNA technology, or chemical synthesis, or anycombination of these or other methods. "Native" or "native sequence"neurotrophic factors or neurotrophins have the amino acid sequence of aneurotrophin occurring in nature in any human or non-human animalspecies, including naturally-occurring truncated and variant forms, andnaturally-occurring allelic variants.

The terms "trk", "trk polypeptide", "trk receptor" and their grammaticalvariants are used interchangeably and refer to polypeptides of thereceptor tyrosine kinase superfamily, which are capable of binding atleast one native neurotrophic factor. Currently identified members ofthis family are trkA (p140^(trkA)), trkB, and trkC, but the definitionspecifically includes polypeptides that might be identified in thefuture as members of this receptor family. The terms "trk", "trkpolypeptide" and "trk receptor", with or without an affixed capitalletter (e.g., A, B or C) designating specific members within thisfamily, specifically include "native" or "native sequence" receptors(wherein these terms are used interchangeably) from any animal species(e.g. human, murine, rabbit, porcine, equine, etc.), including fulllength receptors, their truncated and variant forms, such as thosearising by alternate splicing and/or insertion, and naturally-occurringallelic variants, as well as functional derivatives of such receptors.

Thus, a "native" or "native sequence" human trkB or trkC polypeptide hasthe amino acid sequence of any form of a trkB or trkC receptor asoccurring in the human, including full length native human trkB andtrkC, truncated, tyrosine kinase (TK) domain-deleted (spliced) forms offull length native human trkB and trkC, and insertion variants of fulllength or truncated native human trkC, wherein the insert is within theTK domain or within the extracellular domain, and any furthernaturally-occurring human trkB or trkC polypeptides that might beidentified in the future. A diagram of the different identified forms ofhuman trk polypeptides in comparison to those found in animal species isshown in FIG. 4. Preceded by a signal sequence, the extracellulardomains of full-length native trkA, trkB and trkC receptors have fivefunctional domains, that have been defined with reference to homologousor otherwise similar structures identified in various other proteins(see FIGS. 16A-16C). The domains have been designated starting at theN-terminus of the amino acid sequence of the mature trk receptors as 1)a first cysteine-rich domain extending from amino acid position 1 toabout amino acid position 32 of human trkA, from amino acid position 1to about amino acid position 36 of human trkB, and from amino acidposition 1 to about amino acid position 48 of human trkC; 2) aleucine-rich domain stretching from about amino acid 33 to about aminoacid to about amino acid 104 in trkA; from about amino acid 37 to aboutamino acid 108 in trkB, and from about amino acid 49 to about amino acid120 in trkC; 3) a second cysteine-rich domain from about amino acid 105to about amino acid 157 in trkA; from about amino acid 109 to aboutamino acid 164 in trkB; and from about amino acid 121 to about aminoacid 177 in trkC; 4) a first immunoglobulin-like domain stretching fromabout amino acid 176 to about amino acid 234 in trkA; from about aminoacid 183 to about amino acid 239 in trkB; and from about amino acid 196to about amino acid 257 in trkC; and 5) a second immunoglobulin-likedomain extending from about amino acid 264 to about amino acid 330 intrkA; from about amino acid 270 to about amino acid 334 in trkB; andfrom about amino acid 288 to about amino acid 351 in trkC. The terms"native" or "native sequence" human trkB or trkC specifically includenaturally occurring allelic variants of any native form of thesereceptors. It is noted that the amino acid at position 433 of human trkBwas variously determined to be M or V; both sequences are specificallywithin the scope of the present invention.

A "functional derivative" of a native polypeptide is a compound having aqualitative biological property in common with the native polypeptide. Afunctional derivative of a neurotrophic factor is a compound that has aqualitative biological property in common with a native (human ornon-human) neurotrophic factor. Similarly, a functional derivative of atrk receptor is a compound that has a qualitative biological property incommon with a native (human or non-human) trk receptor. "Functionalderivatives" include, but are not limited to, fragments of nativepolypeptides from any animal species (including humans), and derivativesof native (human and non-human) polypeptides and their fragments,provided that they have a biological activity in common with acorresponding native polypeptide. "Fragments" comprise regions withinthe sequence of a mature native neurotrophic factor or trk receptorpolypeptide. Preferred fragments of trk receptors include at least thesecond immunoglobulin-like domain of a full length native or variant trkreceptor.

The term "derivative" is used to define amino acid sequence andglycosylation variants, and covalent modifications of a nativepolypeptide, whereas the term "variant" refers to amino acid sequenceand glycosylation variants within this definition.

"Biological property" in the context of the definition of "functionalderivatives" is defined as either 1) immunological cross-reactivity withat least one epitope of a native polypeptide (e.g. neurotrophin or trkreceptor), or 2) the possession of at least one adhesive, regulatory oreffector function qualitatively in common with a native polypeptides(e.g. neurotrophin or trk receptor).

Preferably, the functional derivatives are polypeptides which have atleast about 65% amino acid sequence identity, more preferably about 75%amino acid sequence identity, even more preferably at least about 85%amino acid sequence identity, most preferably at least about 95% aminoacid sequence identity with a native polypeptide. In the context of thepresent invention, functional derivatives of native sequence human trkBor trkC polypeptides preferably show at least 95% amino acid sequenceidentity with their cognate native human receptors, and are notimmunogenic in the human, or are fragments of native human trkB or trkCreceptors or of polypeptides exhibiting at least 95% amino acid sequenceidentity with such native receptors, and are not immunogenic in thehuman. The fragments of native full length trk receptors preferablyretain the domain or the domains within the extracellular domain thatare required for ligand binding and/or biological activity. As discussedhereinabove, the extracellular domains of the trk family of proteins arebuild up by five domains: a first cysteine-rich domain, a leucine-richdomain, a second cysteine-rich domain, and two immunoglobulin-likedomains. It is preferred to include in a functional derivative at leastthe second immunoglobulin-like domain of a native trk receptor, or asequence exhibiting at least about 95% sequence identity with the secondimmunoglobulin-like domain of a native trk receptor, wherein the trkreceptor preferably is trkB or trkC.

Amino acid sequence identity or homology is defined herein as thepercentage of amino acid residues in the candidate sequence that areidentical with the residues of a corresponding native polypeptidesequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology, and not consideringany conservative substitutions as part of the sequence identity. NeitherN- or C-terminal extensions nor insertions shall be construed asreducing identity or homology.

Immunologically cross-reactive as used herein means that the candidate(poly)peptide is capable of competitively inhibiting the qualitativebiological activity of a corresponding native polypeptide having thisactivity with polyclonal antibodies or antisera raised against the knownactive molecule. Such antibodies and antisera are prepared inconventional fashion by injecting an animal such as a goat or rabbit,for example, subcutaneously with the known native neurotrophic factor ortrk receptor in complete Feud's adjuvant, followed by boosterintraperitoneal or subcutaneous injection in incomplete Freud's.

"Isolated" nucleic acid or polypeptide in the context of the presentinvention is a nucleic acid or polypeptide that is identified andseparated from contaminant nucleic acids or polypeptides present in theanimal or human source of the nucleic acid or polypeptide. The nucleicacid or polypeptide may be labeled for diagnostic or probe purposes,using a label as described and defined further below in discussion ofdiagnostic assays.

The term "isolated human trkB and trkC polypeptide" and grammaticalvariants thereof refer to human trkB and trkC polypeptides (ashereinabove defined) separated from contaminant polypeptides present inthe human or in other source from which the polypeptide is isolated, andfragments, amino acid sequence variants, glycosylation variants andderivatives of such native sequence polypeptides, provided that theyretain the qualitative ability to bind at least one native neurotrophicfactor, and are not immunogenic in humans. Such isolated human trkB andtrkC polypeptides specifically include native sequence human trkB andtrkC, including the native full-length human trkB and trkC receptors,their naturally-occurring truncated and amino acid (insertion) variantsarising by alternate splicing, and naturally-occurring alleles. Theamino acid sequence variants of native-sequence trkB or trkCpolypeptides show at least about 95% homology, more preferably at leastabout 98% homology with their native counterparts, and arenon-immunogenic to humans. Most preferably, the amino acid sequencevariants within the definition of isolated native human trkB and trkCpolypeptides preserve the entire native sequence of the tyrosine kinasedomain, and the insertions found in naturally-occurring spliced humantrkB or trkC polypeptides. The definition further includes fragments ofthe foregoing native polypeptides and their amino acid sequencevariants, as well as their glycosylation variants and derivativesprovided that they retain the qualitative ability to bind at least onenative neurotrophic factor.

In general, the term "amino acid sequence variant" refers to moleculeswith some differences in their amino acid sequences as compared to areference (e.g. native sequence) polypeptide. The amino acid alterationsmay be substitutions, insertions, deletions or any desired combinationsof such changes in a native amino acid sequence.

Substitutional variants are those that have at least one amino acidresidue in a native sequence removed and a different amino acid insertedin its place at the same position. The substitutions may be single,where only one amino acid in the molecule has been substituted, or theymay be multiple, where two or more amino acids have been substituted inthe same molecule.

Insertional variants are those with one or more amino acids insertedimmediately adjacent to an amino acid at a particular position in anative amino acid sequence. Immediately adjacent to an amino acid meansconnected to either the α-carboxy or α-amino functional group of theamino acid.

Deletional variants are those with one or more amino acids in the nativeamino acid sequence removed. Ordinarily, deletional variants will haveone or two amino acids deleted in a particular region of the molecule.

The term "glycosylation variant" is used to refer to a polypeptidehaving a glycosylation profile different from that of a correspondingnative polypeptide. Glycosylation of polypeptides is typically eitherN-linked or O-linked. N-linked refers to the attachment of thecarbohydrate moiety to the side of an asparagine residue. The tripeptidesequences, asparagine-X-serine and asparagine-X-threonine, wherein X isany amino acid except proline, are recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.O-linked glycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be involved in O-linked glycosylation. Anydifference in the location and/or nature of the carbohydrate moietiespresent in a variant or fragment as compared to its native counterpartis within the scope herein.

The glycosylation pattern of native polypeptides can be determined bywell known techniques of analytical chemistry, including HPAEchromatography Hardy, M.R. et al., Anal. Biochem. 170, 54-62 (1988)!,methylation analysis to determine glycosyl-linkage composition Lindberg,B., Meth. Enzymol. 28. 178-195 (1972); Waeghe, T. J. et al., Carbohydr.Res. 123, 281-304 (1983)!, NMR spectroscopy, mass spectrometry, etc.

"Covalent derivatives" include modifications of a native polypeptide ora fragment thereof with an organic proteinaceous or non-proteinaceousderivatizing agent, and post-translational modifications. Covalentmodifications are traditionally introduced by reacting targeted aminoacid residues with an organic derivatizing agent that is capable ofreacting with selected sides or terminal residues, or by harnessingmechanisms of post-translational modifications that function in selectedrecombinant host cells. Certain post-translational modifications are theresult of the action of recombinant host cells on the expressedpolypeptide. Glutaminyl and asparaginyl residues are frequentlypost-translationally deamidated to the corresponding glutamyl andaspartyl residues. Alternatively, these residues are deamidated undermildly acidic conditions. Either form of these residues may be presentin the trk receptor polypeptides of the present invention. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl, tyrosine orthreonyl residues, methylation of the a-amino groups of lysine,arginine, and histidine side chains T. E. Creighton, Proteins: Structureand Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86(1983)!.

The terms "DNA sequence encoding", "DNA encoding" and "nucleic acidencoding" refer to the order or sequence of deoxyribonucleotides along astrand of deoxyribonucleic acid. The order of these deoxyribonucleotidesdetermines the order of amino acids along the polypeptide chain. The DNAsequence thus codes for the amino acid sequence.

The terms "replicable expression vector" and "expression vecto" refer toa piece of DNA, usually double-stranded, which may have inserted into ita piece of foreign DNA. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell. The vector is used totransport the foreign or heterologous DNA into a suitable host cell.Once in the host cell, the vector can replicate independently of thehost chromosomal DNA, and several copies of the vector and its inserted(foreign) DNA may be generated. In addition, the vector contains thenecessary elements that permit translating the foreign DNA into apolypeptide. Many molecules of the polypeptide encoded by the foreignDNA can thus be rapidly synthesized.

The term "control sequences" refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, a ribosomebinding site, and possibly, other as yet poorly understood sequences.Eukaryotic cells are known to utilize promoters, polyadenylationsignals, and enhancer.

Nucleic acid is "operably linked" when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or a secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, "operably linked"means that the DNA sequences being linked are contiguous and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used in accordwith conventional practice.

In the context of the present invention the expressions "cell", "cellline", and "cell culture" are used interchangeably, and all suchdesignations include progeny. Thus, the words "transformants" and"transformed (host) cells" include the primary subject cell and culturesderived therefrom without regard for the number of transfers. It is alsounderstood that all progeny may not be precisely identical in DNAcontent, due to deliberate or inadvertent mutations. Mutant progeny thathave the same function or biological activity as screened for in theoriginally transformed cell are included. Where distinct designationsare intended, it will be clear from the context.

An "exogenous" element is defined herein to mean nucleic acid sequencethat is foreign to the cell, or homologous to the cell but in a positionwithin the host cell nucleic acid in which the element is ordinarily notfound.

Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having thesame structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V_(H)) followed by a number of constant domains. Eachlight chain has a variable domain at one and (V_(L)) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light and heavy chain variable domains Clothia et al., J.Mol. Biol. 186, 651-663 (1985); Novotny and Haber, Proc. Natl. Acad.Sci. USA 82, 4592-4596 (1985)!.

The variability is not evenly distributed through the variable regionsof antibodies. It is concentrated in three segments calledcomplementarity determining regions (CDRs) or hypervariable regions bothin the light chain and the heavy chain variable regions. The more highlyconserved portions of variable domains are called the framework (FR).The variable domains of native heavy and light chains each comprise fourFR regions, largely adopting a β-sheet configuration, connected by threeCDRs, which form loops connecting, and in some cases forming part of,the β-sheet structure. The CDRs in each chain are held together in closeproximity by the FR regions and, with the CDRs from the other chain,contribute to the formation of the antigen binding site of antibodiessee Kabat, E. A. et al., Sequences of Proteins of Immunological InterestNational Institute of Health, Bethesda, Md. (1987)!. The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen bindingfragments, called Fab fragments, each with a single antigen bindingsite, and a residual "Fc" fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab')₂ fragment thathas two antigen combining sites and is still capable of cross-linkingantigen.

"Fv" is the minimum antibody fragment which contains a complete antigenrecognition and binding site. This region consists of a dimer of oneheavy and one light chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen binding site on thesurface of the V_(H) -V_(L) dimer. Collectively, the six CDRs conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (C_(H) 1) of the heavy chain. Fab'fragments differ from Fab fragments by the addition of a few residues atthe carboxy terminus of the heavy chain C_(H) 1 domain including one ormore cysteines from the antibody hinge region. Fab'-SH is thedesignation herein for Fab' in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab')₂ antibody fragmentsoriginally were produced as pairs of Fab' fragments which have hingecysteines between them. Other, chemical couplings of antibody fragmentsare also known.

The light chains of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa and lambda (λ), based on the amino acid sequences of theirconstant domains.

Depending on the amino acid sequence of the constant region of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG andIgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. Theheavy chain constant regions that correspond to the different classes ofimmunoglobulins are called α, delta, epsilon, γ, and μ, respectively.The subunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known. IgA-1 and IgA-2 are monomericsubclasses of IgA, which usually is in the form of dimers or largerpolymers. Immunocytes in the gut produce mainly polymeric IgA (alsoreferred to poly-IgA including dimers and higher polymers). Suchpoly-IgA contains a disulfide-linked polypeptide called the "joining" or"J" chain, and can be transported through the glandular epitheliumtogether with the J-containing polymeric IgM (poly-IgM), comprising fivesubunits.

The term "antibody" is used in the broadest sense and specificallycovers single anti-trk monoclonal antibodies (including agonist andantagonist antibodies) and anti-trk antibody compositions withpolyepitopic specificity.

The term "monoclonal antibody" as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins.

The monoclonal antibodies herein include hybrid and recombinantantibodies produced by splicing a variable (including hypervariable)domain of an anti-trk antibody with a constant domain (e.g. "humanized"antibodies), or a light chain with a heavy chain, or a chain from onespecies with a chain from another species, or fusions with heterologousproteins, regardless of species of origin or iimunoglobulin class orsubclass designation, as well as antibody fragments (e.g., Fab, F(ab')₂,and Fv), so long as they exhibit the desired biological activity. See,e.g. Cabilly, et al., U.S. Pat. No. 4,816,567; Mage & Lamoyi, inMonoclonal Antibody Production Techniques and Applications, pp. 79-97(Marcel Dekker, Inc., New York, 1987).!

Thus, the modifier "monoclonal" indicates the character of the antibodyas being obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler & Milstein,Nature 256:495 (1975), or may be made by recombinant DNA methodsCabilly, et al., supra!.

"Humanized" forms of non-human (e.g. murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (PR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibody may comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Hybridization is preferably performed under "stringent conditions" whichmeans (1) employing low ionic strength and high temperature for washing,for example, 0.015 sodium chloride/0.0015 M sodium citrate/0.1% sodiumdodecyl sulfate at 50° C., or (2) employing during hybridization adenaturing agent, such as formamide, for example, 50% (vol/vol)formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 nM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C. Another example is useof 50% formamide, 5 x SSC (0.75M NaCl, 0.075M sodium citrate), 50 mMsodium phosphate (pH 6/8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2× SSC and 0.1%SDS.

B. Isolation of DNA Encoding the Term Receptors

For the purpose of the present invention, DNA encoding a trk receptorcan be obtained from any cDNA library prepared from tissue believed topossess the trk receptor MRNA and to express it at a detectable level.For example, a human brain cDNA library, such as that described in theexamples, is a good source of trkB and trkC receptor cDNA. The trkreceptor genes can also be obtained from a genomic library, such as ahuman genomic cosmic library.

Identification of trk receptor DNA is most conveniently accomplished byprobing human or other mammalian cDNA or genomic libraries by labeledoligonucleotide sequences selected from known trk sequences (such ashuman trkA sequence, murine trkB sequence or murine or porcine trkCsequence) in accord with known criteria, among which is that thesequence should be sufficient in length and sufficiently unambiguousthat false positives are minimized. Typically, a ³² P-labeledoligonucleotide having about 30 to 50 bases is sufficient, particularlyif the oligonucleotide contains one or more codons for methionine ortryptophan. Isolated nucleic acid will be DNA that is identified andseparated from contaminant nucleic acid encoding other polypeptides fromthe source of nucleic acid.

An alternative means to isolate the gene encoding a trk receptor is touse polymerase chain reaction (PCR) methodology as described in U.S.Pat. No. 4,683,195, issued Jul. 28, 1987, in section 14 of Sambrook etal., Molecular Cloning: A Laboratory Manual, second edition, Cold SpringHarbor Laboratory Press. New York, 1989, or in Chapter 15 of CurrentProtocols in Molecular Biology, Ausubel et al. eds., Greene PublishingAssociates and Wiley-Interscience 1991, and as illustrated in theexamples.

Another alternative is to chemically synthesize the gene encoding a trkreceptor, using one of the methods described in Engels and Uhlmann,Agnew. Chem. Int. Ed. Engl. 2, 716 (1989). These methods includetriester, phosphite, phosphoramidite and H-phosphonate methods, PCR andother autoprimer methods, and oligonucleotide syntheses on solidsupports.

C. Amino Acid Sequence Variants of a Native trk Receptor or ReceptorFragments

Amino acid sequence variants of native trk receptors and trk receptorfragments are prepared by methods known in the art by introducingappropriate nucleotide changes into a native or variant trk receptorDNA, or by in vitro synthesis of the desired polypeptide. There are twoprincipal variables in the construction of amino acid sequence variants:the location of the mutation site and the nature of the mutation. Withthe exception of naturally-occurring alleles, which do not require themanipulation of the DNA sequence encoding the trk receptor, the aminoacid sequence variants of trk receptor are preferably constructed bymutating the DNA, either to arrive at an allele or an amino acidsequence variant that does not occur in nature. In general, themutations will be created within the extracellular domain of a nativetrk receptor. Sites or regions that appear to be important for thesignal transduction of a neurotrophic factor, will be selected in invitro studies of neurotrophin biological activity. Sites at suchlocations will then be modified in series, e.g. by (1) substitutingfirst with conservative choices and then with more radical selectionsdepending upon the results achieved, (2) deleting the target residue orresidues, or (3) inserting residues of the same or different classadjacent to the located site, or combinations of options 1-3.

One helpful technique is called "alanine scanning" (Cunningham andWells, Science 244, 1081-1085 1989!). Here, a residue or group of targetresidues is identified and substituted by alanine or polyalanine. Thosedomains demonstrating functional sensitivity to the alaninesubstitutions are then refined by introducing further or othersubstituents at or for the sites of alanine substitution.

After identifying the desired mutation(s), the gene encoding a trkreceptor variant can be obtained by chemical synthesis as hereinabovedescribed.

More preferably, DNA encoding an trk receptor amino acid sequencevariant is prepared by site-directed mutagenesis of DNA that encodes anearlier prepared variant or a nonvariant version of trk receptor.Site-directed (site-specific) mutagenesis allows the production of trkreceptor variants through the use of specific oligonucleotide sequencesthat encode the DNA sequence of the desired mutation, as well as asufficient number of adjacent nucleotides, to provide a primer sequenceof sufficient size and sequence complexity to form a stable duplex onboth sides of the deletion junction being traversed. Typically, a primerof about 20 to 25 nucleotides in length is preferred, with about 5 to 10residues on both sides of the junction of the sequence being altered. Ingeneral, the techniques of site-specific mutagenesis are well known inthe art, as exemplified by publications such as, Edelman et al., DNA 2,183 (1983). As will be appreciated, the site-specific mutagenesistechnique typically employs a phage vector that exists in both asingle-stranded and double-stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage, forexample, as disclosed by Messing et al., Third Cleveland Symposium onMacromolecules and Recombinant DNA, A. Walton, ed., Elsevier, Amsterdam(1981). This and other phage vectors are commercially available andtheir use is well known to those skilled in the art. A versatile andefficient procedure for the construction of oligodeoxyribonucleotidedirected site-specific mutations in DNA fragments using M13-derivedvectors was published by Zoller, M. J. and Smith, M., Nucleic Acids Res.10, 6487-6500 1982!). Also, plasmid vectors that contain asingle-stranded phage origin of replication (Veira et al., Meth.Enzymol. 153, 3 1987!) may be employed to obtain single-stranded DNA.Alternatively, nucleotide substitutions are introduced by synthesizingthe appropriate DNA fragment in vitro, and amplifying it by PCRprocedures known in the art.

In general, site-specific mutagenesis herewith is performed by firstobtaining a single-stranded vector that includes within its sequence aDNA sequence that encodes the relevant protein. An oligonucleotideprimer bearing the desired mutated sequence is prepared, generallysynthetically, for example, by the method of Crea et al., Proc. Natl.Acad. Sci. USA 75, 5765 (1978). This primer is then annealed with thesingle-stranded protein sequence-containing vector, and subjected toDNA-polymerizing enzymes such as, E. coli polymerase I Klenow fragment,to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate hostcells such as JP101 cells, and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.Thereafter, the mutated region may be removed and placed in anappropriate expression vector for protein production.

The PCR technique may also be used in creating amino acid sequencevariants of a trk receptor. When small amounts of template DNA are usedas starting material in a PCR, primers that differ slightly in sequencefrom the corresponding region in a template DNA can be used to generaterelatively large quantities of a specific DNA fragment that differs fromthe template sequence only at the positions where the primers differfrom the template. For introduction of a mutation into a plasmid DNA,one of the primers is designed to overlap the position of the mutationand to contain the mutation; the sequence of the other primer must beidentical to a stretch of sequence of the opposite strand of theplasmid, but this sequence can be located anywhere along the plasmidDNA. It is preferred, however, that the sequence of the second primer islocated within 200 nucleotides from that of the first, such that in theend the entire amplified region of DNA bounded by the primers can beeasily sequenced. PCR amplification using a primer pair like the onejust described results in a population of DNA fragments that differ atthe position of the mutation specified by the primer, and possibly atother positions, as template copying is somewhat error-prone.

If the ratio of template to product material is extremely low, the vastmajority of product DNA fragments incorporate the desired mutation(s).This product material is used to replace the corresponding region in theplasmid that served as PCR template using standard DNA technology.Mutations at separate positions can be introduced simultaneously byeither using a mutant second primer or performing a second PCR withdifferent mutant primers and ligating the two resulting PCR fragmentssimultaneously to the vector fragment in a three (or more) partligation.

In a specific example of PCR mutagenesis, template plasmid DNA (1 μg) islinearized by digestion with a restriction endonuclease that has aunique recognition site in the plasmid DNA outside of the region to beamplified. Of this material, 100 ng is added to a PCR mixture containingPCR buffer, which contains the four deoxynucleotide triphosphates and isincluded in the GeneAmp^(R) kits (obtained from Perkin-Elmer Cetus,Norwalk, Conn. and Emeryville, Calif.), and 25 pmole of eacholigonucleotide primer, to a final volume of 50 μl. The reaction mixtureis overlayered with 35 μl mineral oil. The reaction is denatured for 5minutes at 100° C., placed briefly on ice, and then 1 μl Thermusaquaticus (Taq) DNA polymerase (5 units/ 1), purchased from Perkin-ElmerCetus, Norwalk, Conn. and Emeryville, Calif.) is added below the mineraloil layer. The reaction mixture is then inserted into a DNA ThermalCycler (purchased from Perkin-Elmer Cetus) programmed as follows:

2 min. 55° C.,

30 sec. 72° C., then 19 cycles of the following:

30 sec. 94° C.,

30 sec. 55° C., and

30 sec. 72° C.

At the end of the program, the reaction vial is removed from the thermalcycler and the aqueous phase transferred to a new vial, extracted withphenol/chloroform (50:50 vol), and ethanol precipitated, and the DNA isrecovered by standard procedures. This material is subsequentlysubjected to appropriate treatments for insertion into a vector.

Another method for preparing variants, cassette mutagenesis, is based onthe technique described by Wells et al. Gene 34, 315 (1985)!. Thestarting material is the plasmid (or vector) comprising the trk receptorDNA to be mutated. The codon(s) within the trk receptor to be mutatedare identified. There must be a unique restriction endonuclease site oneach side of the identified mutation site(s). If no such restrictionsites exist, they may be generated using the above-describedoligonucleotide-mediated mutagenesis method to introduce them atappropriate locations in the trk receptor DNA. After the restrictionsites have been introduced into the plasmid, the plasmid is cut at thesesites to linearize it. A double-stranded oligonucleotide encoding thesequence of the DNA between the restriction site but containing thedesired mutation(s) is synthesized using standard procedures. The twostrands are synthesized separately and then hybridized together usingstandard techniques. This double-stranded oligonucleotide is referred toas the cassette. This cassette is designed to have 3' and 5' ends thatare compatible with the ends of the linearized plasmid, such that it canbe directly ligated to the plasmid. This plasmid now contains themutated trk receptor DNA sequence.

Additionally, the so-called phagemid display method may be useful inmaking amino acid sequence variants of native or variant trk receptorsor their fragments. This method involves (a) constructing a replicableexpression vector comprising a first gene encoding an receptor to bemutated, a second gene encoding at least a portion of a natural orwild-type phage coat protein wherein the first and second genes areheterologous, and a transcription regulatory element operably linked tothe first and second genes, thereby forming a gene fusion encoding afusion protein; (b) mutating the vector at one or more selectedpositions within the first gene thereby forming a family of relatedplasmids; (c) transforming suitable host cells with the plasmids; (d)infecting the transformed host cells with a helper phage having a geneencoding the phage coat protein; (e) culturing the transformed infectedhost cells under conditions suitable for forming recombinant phagemidparticles containing at least a portion of the plasmid and capable oftransforming the host, the conditions adjusted so that no more than aminor amount of phagemid particles display more than one copy of thefusion protein on the surface of the particle; (f) contacting thephagemid particles with a suitable antigen so that at least a portion ofthe phagemid particles bind to the antigen; and (g) separating thephagemid particles that bind from those that do not. Steps (d) through(g) can be repeated one or more times. Preferably in this method theplasmid is under tight control of the transcription regulatory element,and the culturing conditions are adjusted so that the amount or numberof phagemid particles displaying more than one copy of the fusionprotein on the surface of the particle is less than about 1%. Also,preferably, the amount of phagemid particles displaying more than onecopy of the fusion protein is less than 10% of the amount of phagemidparticles displaying a single copy of the fusion protein. Mostpreferably, the amount is less than 20%. Typically in this method, theexpression vector will further contain a secretory signal sequence fusedto the DNA encoding each subunit of the polypeptide and thetranscription regulatory element will be a promoter system. Preferredpromoter systems are selected from lac Z, λ_(PL), tac, T7 polymerase,tryptophan, and alkaline phosphatase promoters and combinations thereof.Also, normally the method will employ a helper phage selected fromM13K07, M13R408, M13-VCS, and Phi X 174. The preferred helper phage isM13K07, and the preferred coat protein is the M13 Phage gene III coatprotein. The preferred host is E. coli, and protease-deficient strainsof E. coli.

Further details of the foregoing and similar mutagenesis techniques arefound in general textbooks, such as, for example, Sambrook et al.,supra, and Current Protocols in Molecular Biology, Ausubel et al. eds.,supra.

Amino acid substitution variants have at least one amino acid residue ina native receptor molecule removed and a different residue inserted inits place. The sites of great interest for substitutional mutagenesisinclude sites identified as important for signal transduction and/orligand binding, and sites where the amino acids found in the native trkreceptors from various species are substantially different in terms ofside bulk, charge and/or hydrophobicity. As it will be apparent from theexamples, the second immunoglobulin-like domain of the human trkCreceptor has been identified as primarily responsible for neurotrophinbinding. Substitutions (just as other amino acid alterations) withinthis region are believed to significantly affect the neurotrophinbinding properties of trk receptors. Amino acid(s) primarily responsiblefor the binding specificity of and the diverse biological activitiesmediated by the individual trk receptors can be identified by acombination of the foregoing mutagenesis techniques. At least part ofthe amino acids distinguishing the various trk receptors from oneanother are believed to be within the second immunoglobulin-like domainof their extracellular region. It is possible to create trk receptorvariants by substituting the region identified as responsible forligand-specificity in one trk receptor by the ligand binding domain ofanother trk receptor.

Other sites of interest are those in which particular residues of thenative trk receptors from various species are identical. These positionsmay be important for the biological function of the trk receptor.Further important sites for mutagenesis include motifs common in variousmembers of the trk receptor family.

Naturally-occurring amino acids are divided into groups based on commonside chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophobic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Conservative substitutions involve exchanging a member within one groupfor another member within the same group, whereas non-conservativesubstitutions will entail exchanging a member of one of these classesfor another. Variants obtained by non-conservative substitutions withinthe neurotrophic factor-binding region(s) of a native trk receptorsequence of a fragment thereof are expected to result in signif icantchanges in the biological properties of the obtained variant, and mayresult in trk receptor variants which block the biological activity oftheir cognate neurotrophic factor(s), i.e. are antagonists of thebiological action of the corresponding native neurotrophic factor(s), orthe signaling potential of which surpasses that of the correspondingnative trk receptor. Amino acid positions that are conserved amongvarious species and/or various receptors of the trk receptor family aregenerally substituted in a relatively conservative manner if the goal isto retain biological activity.

Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Deletions may be introduced into regions not directlyinvolved in signal transduction and/or ligand binding, to modify thebiological activity of the trk receptor. Deletions from the regions thatare directly involved in signal transduction and/or ligand binding willbe more likely to modify the biological activity of the mutated trkreceptor more significantly, and may potentially yield trk receptorantagonists. The number of consecutive deletions will be selected so asto preserve the tertiary structure of the trk receptor in the affecteddomain.

It is possible to construct trk receptor variants which combine thebinding domains for and, accordingly, have the ability to signal thebiological activities of more than one neurotrophic factor. Such variantcan be made by inserting into the sequence of a trk receptor theneurotrophin binding domain of another trk receptor. For example, nativetrkB and trkC receptors do not bind to an appreciable degree NGF, whichis the native ligand for the trkA receptor. Insertion of the NGF-bindingsequence of a trkA receptor into a trkB or trkC receptor yields a trkBor trkC receptor variant, which (in addition to the native ligands ofthe native trkB and trkC receptors, respectively) binds NGF. Similarly,naturally occurring trkB receptors bind BDNF and NT4/5 but do not bindappreciably to NGF or NT-3. Thus, the insertion of the NT-3 bindingsequence of trkC into a trkB receptor yields a variant receptor that iscapable of binding BDNF, NT4/5 and NT-3. The resultant receptor variantswill be able to mediate a broader spectrum of biological activities,which opens new ways for their application and therapeutics.

Amino acid insertions also include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Intrasequence insertions (i.e.insertions within the trk receptor amino acid sequence) may rangegenerally from about 1 to 10 residues, more preferably 1 to 5 residues,more preferably 1 to 3 residues. Examples of terminal insertions includethe trk receptor with an N-terminal methionyl residue, an artifact ofits direct expression in bacterial recombinant cell culture, and fusionof a heterologous N-terminal signal sequence to the N-terminus of thetrk receptor molecule to facilitate the secretion of the mature trkreceptor from recombinant host cells. Such signal sequences willgenerally be obtained from, and thus homologous to, the intended hostcell species. Suitable sequences include STII or Ipp for E. coli, alphafactor for yeast, and viral signals such as herpes gD for mammaliancells.

Other insertional variants of the native trk receptor molecules includethe fusion to the N- or C-terminus of the trk receptor of immunogenicpolypeptides, e.g. bacterial polypeptides such as beta-lactamase or anenzyme encoded by the E. coli trp locus, or yeast protein, andC-terminal fusions with proteins having a long half-life such asimmunoglobulin regions (preferably immunoglobulin constant regions),albumin, or ferritin, as described in WO 89/02922 published on Apr. 6,1989.

Since it is often difficult to predict in advance the characteristics ofa variant trk receptor, it will be appreciated that some screening willbe needed to select the optimum variant.

D. Insertion of DNA into a Cloning Vehicle

Once the nucleic acid encoding a native or variant trk receptor isavailable, it is generally ligated into a replicable expression vectorfor further cloning (amplification of the DNA), or for expression.

Expression and cloning vectors are well known in the art and contain anucleic acid sequence that enables the vector to replicate in one ormore selected host cells. The selection of the appropriate vector willdepend on 1) whether it is to be used for DNA amplification or for DNAexpression, 2) the size of the DNA to be inserted into the vector, and3) the host cell to be transformed with the vector. Each vector containsvarious components depending on its function (amplification of DNA ofexpression of DNA) and the host cell for which it is compatible. Thevector components generally include, but are not limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(i) Signal Sequence Component

In general, the signal sequence may be a component of the vector, or itmay be a part of the trk receptor that is inserted into the vector. Thenative trk receptor comprises a signal sequence at the amino terminus(5' end of the DNA) of the polypeptide that is cleaved duringpost-translational processing of the polypeptide to form a mature trkreceptor. Native trk receptor is however not secreted from the host cellas it contains a membrane anchoring domain between the extracellulardomain and the cytoplasmic domain Thus, to form a secreted version of antrk receptor, the membrane anchoring domain (also referred to astransmembrane domain) is ordinarily deleted or otherwise inactivated(for example by point mutation(s)). Generally, the cytoplasmic domain isalso deleted along with the membrane anchoring domain. The truncated (ortransmembrane domain-inactivated) trk receptor variants may be secretedfrom the cell, provided that the DNA encoding the truncated variantretains the amino terminal signal sequence.

Included within the scope of this invention are trk receptors with thenative signal sequence deleted and replaced with a heterologous signalsequence. The heterologous signal sequence selected should be one thatis recognized and processed (i.e. cleaved by a signal peptidase) by thehost cell.

For prokaryotic host cells that do not recognize and process the nativetrk receptor signal sequence, the signal sequence is substituted by aprokaryotic signal sequence selected, for example, from the group of thealkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin IIleaders. For yeast secretion the native trk receptor signal sequence maybe substituted by the yeast invertase, alpha factor, or acid phosphataseleaders. In mammalian cell expression the native signal sequence issatisfactory, although other mammalian signal sequences may be suitable.

(ii) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenabled the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomes, and includesorigins of replication or autonomously replicating sequences. Suchsequence are well known for a variety of bacteria, yeast and viruses.The origin of replication from the well-known plasmid pBR322 is suitablefor most gram negative bacteria, the 2μ plasmid origin for yeast andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells. Origins of replication are notneeded for mammalian expression vectors (the SV40 origin may typicallybe used only because it contains the early promoter). Most expressionvectors are "shuttle" vectors, i.e. they are capable of replication inat least one class of organisms but can be transfected into anotherorganism for expression. For example, a vector is cloned in E. coli andthen the same vector is transfected into yeast or mammalian cells forexpression even though it is not capable of replicating independently ofthe host cell chromosome.

DNA is also cloned by insertion into the host genome. This is readilyaccomplished using Bacillus species as hosts, for example, by includingin the vector a DNA sequence that is complementary to a sequence foundin Bacillus genomic DNA. Transfection of Bacillus with this vectorresults in homologous recombination with the genome and insertion of theDNA encoding the desired heterologous polypeptide. However, the recoveryof genomic DNA is more complex than that of an exogenously replicatedvector because restriction enzyme digestion is required to excise theencoded polypeptide molecule.

(iii) Selection Gene Component

Expression and cloning vectors should contain a selection gene, alsotermed a selectable marker. This is a gene that encodes a proteinnecessary for the survival or growth of a host cell transformed with thevector. The presence of this gene ensures that any host cell whichdeletes the vector will not obtain an advantage in growth orreproduction over transformed hosts. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins, e.g.ampicillin, neomycin, methotrexate or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g. the gene encoding D-alanine racemase forbacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene express a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin Southern et al., J. Molec. Appl. Genet. 1, 327(1982)!, mycophenolic acid Mulligan et al., Science 209, 1422 (1980)!,or hygromycin Sudgen et al., Mol. Cel. Biol. 5, 410-413 (1985)!. Thethree examples given above employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

Other examples of suitable selectable markers for mammalian cells aredihydrofolate reductase (DHFR) or thymidine kinase. Such markers enablethe identification of cells which were competent to take up the desirednucleic acid. The mammalian cell transformants are placed underselection pressure which only the transformants are uniquely adapted tosurvive by virtue of having taken up the marker. Selection pressure isimposed by culturing the transformants under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to amplification of both the selection gene and the DNAthat encodes the desired polypeptide. Amplification is the process bywhich genes in greater demand for the production of a protein criticalfor growth are reiterated in tandem within the chromosomes of successivegenerations of recombinant cells. Increased quantities of the desiredpolypeptide (either a trk-containing chimeric polypeptide or a segmentthereof) are synthesized from the amplified DNA.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumwhich lacks hypoxanthine, glycine, and thymidine. An appropriate hostcell in this case is the Chinese hamster ovary (CHO) cell line deficientin DHFR activity, prepared and propagated as described by Urlaub andChasin, Proc. Nat'l. Acad. Sci. USA 77, 4216 (1980). A particularlyuseful DHFR is a mutant DHFR that is highly resistant to MTX (EP117,060). This selection agent can be used with any otherwise suitablehost, e.g. ATCC No. CCL61 CHO-K1, notwithstanding the presence ofendogenous DHFR. The DNA encoding DHFR and the desired polypeptide,respectively, then is amplified by exposure to an agent (methotrexate,or MTX) that inactivates the DHFR. One ensures that the cell requiresmore DHFR (and consequently amplifies all exogenous DNA) by selectingonly for cells that can grow in successive rounds of ever-greater MTXconcentration. Alternatively, hosts co-transformed with genes encodingthe desired polypeptide, wild-type DHFR, and another selectable markersuch as the neo gene can be identified using a selection agent for theselectable marker such as G418 and then selected and amplified usingmethotrexate in a wild-type host that contains endogenous DHFR. (Seealso U.S. Pat. No. 4,965,199).

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature 282:39; Kingsmanet al., 1979, Gene 7:141; or Tschemper et al., 1980, Gene 10:157). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1 (Jones, 1977, Genetics 85:12). The presence of the trp1 lesionin the yeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.Similarly, Leu2 deficient yeast strains (ATCC 20,622 or 38,626) arecomplemented by known plasmids bearing the Leu2 gene.

(iv) Promoter Component

Expression vectors, unlike cloning vectors, should contain a promoterwhich is recognized by the host organism and is operably linked to thenucleic acid encoding the desired polypeptide. Promoters areuntranslated sequences located upstream from the start codon of astructural gene (generally within about 100 to 1000 bp) that control thetranscription and translation of nucleic acid under their control. Theytypically fall into two classes, inducible and constitutive. Induciblepromoters are promoters that initiate increased levels of transcriptionfrom DNA under their control in response to some change in cultureconditions, e.g. the presence or absence of a nutrient or a change intemperature. At this time a large number of promoters recognized by avariety of potential host cells are well known. These promoters areoperably linked to DNA encoding the desired polypeptide by removing themfrom their gene of origin by restriction enzyme digestion, followed byinsertion 5' to the start codon for the polypeptide to be expressed.This is not to say that the genomic promoter for trk receptor is notusable. However, heterologous promoters generally will result in greatertranscription and higher yields of expressed trk receptor as compared tothe native trk receptor promoter.

Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., Nature 275:615(1978); and Goeddel et al., Nature 281:544 (1979)), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes. 8:4057 (1980) and EPO Appln. Publ. No. 36,776) and hybrid promoterssuch as the tac promoter (H. de Boer et al., Proc. Nat'l. Acad. Sci. USA80:21-25 (1983)). However, other known bacterial promoters are suitable.Their nucleotide sequences have been published, thereby enabling askilled worker operably to ligate them to DNA encoding trk (Siebenlistet al., Cell 2:269 (1980)) using linkers or adaptors to supply anyrequired restriction sites. Promoters for use in bacterial systems alsowill contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding trk.

Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al. J. Biol. Chem.255:2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149 (1978); and Holland, Biochemistry 17:4900 (1978)),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phospho-fructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin R. Hitzeman et al., EP 73,657A. Yeast enhancers also areadvantageously used with yeast promoters.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CXCAAT region where X may be any nucleotide. At the3' end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3' end of the codingsequence. All of these sequences are suitably inserted into mammalianexpression vectors.

trk receptor transcription from vectors in mammalian host cells may becontrolled by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus (UK 2,211,504 published Jul. 5, 1989),adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, cytomegalovirus, a retrovirus, hepatitis-B virus and mostpreferably Simian Virus 40 (SV40), from heterologous mammalianpromoters, e.g. the actin promoter or an immunoglobulin promoter, fromheat shock promoters, and from the promoter normally associated with thetrk receptor sequence, provided such promoters are compatible with thehost cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment which also contains the SV40 viralorigin of replication Fiers et al., Nature 273:113 (1978), Mulligan andBerg, Science 209, 1422-1427 (1980); Pavlakis et al., Proc. Natl. Acad.Sci. USA 78, 7398-7402 (1981)!. The immediate early promoter of thehuman cytomegalovirus is conveniently obtained as a HindIII Erestriction fragment Greenaway et al., Gene 18, 355-360 (1982)!. Asystem for expressing DNA in mammalian hosts using the bovine papillomavirus as a vector is disclosed in U.S. Pat. No. 4,419,446. Amodification of this system is described in U.S. Pat. No. 4,601,978. Seealso, Gray et al., Nature 295, 503-508 (1982) on expressing cDNAencoding human immune interferon in monkey cells; Reyes et al., Nature297, 598-601 (1982) on expressing human β-interferon cDNA in mouse cellsunder the control of a thymidine kinase promoter from herpes simplexvirus; Canaani and Berg, Proc. Natl. Acad. Sci. USA 79, 5166-5170 (1982)on expression of the human interferon β1 gene in cultured mouse andrabbit cells; and Gorman et al., Proc. Natl. Acad. Sci. USA 79,6777-6781 (1982) on expression of bacterial CAT sequences in CV-1 monkeykidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells,HeLa cells, and mouse HIN-3T3 cells using the Rous sarcoma virus longterminal repeat as a promoter.

The actual plasmid used in the course of cloning the murine trk receptorcontains the promoter of the murine 3-hydroxy-3-methylglutaryl coenzymeA reductase gene Gautier et al., Nucleic Acids Res. 17, 8389 (1989)!,whereas the reporter plasmid pUMS (GT)₈ -Tac! used during expressioncloning contained an artificial multimerized trk recepto-induciblepromoter element McDonald et al., Cell 60, 767-779 (1990)!.

(v) Enhancer Element Component

Transcription of a DNA encoding the trk receptors of the presentinvention by higher eukaryotes is often increased by inserting anenhancer sequence into the vector. Enhancers are cis-acting elements ofDNA, usually about from 10 to 300 bp, that act on a promoter to increaseits transcription. Enhancers are relatively orientation and positionindependent having been found 5' Laimins et al., Proc. Natl. Acad. Sci.USA 78, 993 (1981)! and 3' Lasky et al., Mol Cel. Biol. 3, 1108 (1983)!to the transcription unit, within an intron Banerji et al., Cell 33, 729(1983)! as well as within the coding sequence itself Osborne et al.,Mol. Cel. Biol. 4, 1293 (1984)!. Many enhancer sequences are now knownfrom mammalian genes (globin, elastase, albumin, α-fetoprotein andinsulin). Typically, however, one will use an enhancer from a eukaryoticcell virus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297, 17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5' or 3' to thetrk receptor DNA, but is preferably located at a site 5' from thepromoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5' and, occasionally 3' untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the trk receptor. The 3' untranslatedregions also include transcription termination sites.

Construction of suitable vectors containing one or more of the abovelisted components, the desired coding and control sequences, employsstandard ligation techniques. Isolated plasmids or DNA fragments arecleaved, tailored, and religated in the form desired to generate theplasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) and successful transformants selected by ampicillin ortetracycline resistance where appropriate. Plasmids from thetransformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Messing et al., NucleicAcids Res. 9, 309 (1981) or by the method of Maxam et al., Methods inEnzymology 65, 499 (1980).

Particularly useful in the practice of this invention are expressionvectors that provide for the transient expression in mammalian cells ofDNA encoding an trk receptor. In general, transient expression involvesthe use of an expression vector that is able to replicate efficiently ina host cell, such that the host cell accumulates many copies of theexpression vector and, in turn, synthesizes high levels of a desiredpolypeptide encoded by the expression vector. Transient systems,comprising a suitable expression vector and a host cell, allow for theconvenient positive identification of polypeptides encoded by clonesDNAs, as well as for the rapid screening of such polypeptides fordesired biological or physiological properties. Thus, transientexpression systems are particularly useful in the invention for purposesof identifying analogs and variants of the trk receptor.

Other methods, vectors, and host cells suitable for adaptation to thesynthesis of the trk receptors in recombinant vertebrate cell cultureare described in Getting et al., Nature 293, 620-625 (1981); Mantel etal., Nature 281, 40-46 (1979); Levinson et al.; EP 117,060 and EP117,058. A particularly useful plasmid for mammalian cell cultureexpression of the trk receptor is pRK5 (EP 307,247).

E. Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the vectors herein are theprokaryote, yeast or higher eukaryote cells described above. Suitableprokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. A preferred cloning host is E. coli 294(ATCC 31,446) although other gram negative or gram positive prokaryotessuch as E. coli B, E. coli X1776 (ATCC 31,537), E. coli W3110 (ATCC27,325), Pseudomonas species, or Serratia Marcesans are suitable.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable hosts for vectors herein. Saccharomycescerevisiae, or common baker's yeast, is the most commonly used amonglower eukaryotic host microorganisms. However, a number of other genera,species and strains are commonly available and useful herein, such as S.pombe Beach and Nurse, Nature 290, 140 (1981)!, Kluyveromyces lactisLouvencourt et al., J. Bacteriol. 737 (1983)!; yarrowia (EP 402,226);Pichia pastoris (EP 183,070), Trichoderma reesia (EP 244,234),Neurospora crassa Case et al., Proc. Natl. Acad. Sci. USA 76, 5259-5263(1979)!; and Aspergillus hosts such as A. nidulans Ballance et al.,Biochem. Biophys. Res. Commun. 112, 284-289 (1983); Tilburn et al., Gene12, 205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA 81,1470-1474 (1984)! and A. niger Kelly and Hynes, EMBO J. 4, 475-479(1985)!.

Suitable host cells may also derive from multicellular organisms. Suchhost cells are capable of complex processing and glycosylationactivities. In principle, any higher eukaryotic cell culture isworkable, whether from vertebrate or invertebrate culture, althoughcells from mammals such as humans are preferred. Examples ofinvertebrate cells include plants and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegyti(mosquito), Aedes albopictus (mosquito), Drosophila melangaster(fruitfly), and Bombyx mori host cells have been identified. See, e.g.Luckow et al., Bio/Technology 6, 47-55 (1988); Miller et al., in GeneticEngineering, Setlow, J. K. et al., eds., Vol. 8 (Plenum Publishing,1986), pp. 277-279; and Maeda et al., Nature 315, 592-594 (1985). Avariety of such viral strains are publicly available, e.g. the L-1variant of Autographa californica NPV, and such viruses may be used asthe virus herein according to the present invention, particularly fortransfection of Spodoptera fruaiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,and tobacco can be utilized as hosts. Typically, plant cells aretransfected by incubation with certain strains of the bacteriumAgrobacterium tumefaciens, which has been previously manipulated tocontain the trk receptor DNA. During incubation of the plant cellculture with A. tumefaciens, the DNA encoding trk receptor istransferred to the plant cell host such that it is transfected, andwill, under appropriate conditions, express the trk receptor DNA. Inaddition, regulatory and signal sequences compatible with plant cellsare available, such as the nopaline synthase promoter andpolyadhenylation signal sequences. Depicker et al., J. Mol. Appl. Gen.1, 561 (1982). In addition, DNA segments isolated from the upstreamregion of the T-DNA 780 gene are capable of activating or increasingtranscription levels of plant-expressible genes in recombinantDNA-containing plant tissue. See EP 321,196 published Jun. 21, 1989.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) is per se well known.See Tissue Culture, Academic Press, Kruse and Patterson, editors (1973).Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cellline 293 or 293 cells subcloned for growth in suspension culture, Grahamet al., J. Gen. Virol. 36, 59 (1977)!; baby hamster kidney cells 9BHK,ATCC CCL 10); Chinese hamster ovary cells/-DHFR CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA 77, 4216 (1980)!; mouse sertolli cells ITM4,Mather, Biol. Reprod. 23, 243-251 (1980)!; monkey kidney cells (CV1 ATCCCCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442);human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells Mather etal., Annals N.Y. Acad. Sci. 383, 44068 (1982)!; MRC 5 cells; FS4 cells;and a human hepatoma cell line (Hep G2). Preferred host cells are humanembryonic kidney 293 and Chinese hamster ovary cells.

Particularly preferred host cells for the purpose of the presentinvention are vertebrate cells producing the trk receptor.

Host cells are transfected and preferably transformed with theabove-described expression or cloning vectors and cultured inconventional nutrient media modified as is appropriate for inducingpromoters or selecting transformants containing amplified genes.

F. Culturing the Host Cells

Prokaryotes cells used to produced the trk receptor polypeptides of thisinvention are cultured in suitable media as describe generally inSambrook et al., supra.

Mammalian cells can be cultured in a variety of media. Commerciallyavailable media such as Ham's F10 (Sigma), Minimal Essential Medium(MEM, Sigma), RPMI-1640)Sigma), and Dulbecco's Modified Eagle's Medium(DMEM, Sigma) are suitable for culturing the host cells. In addition,any of the media described in Ham and Wallace, Meth. Enzymol. 58, 44(1979); Barnes and Sato, Anal, Biochem. 102, 255 (1980), U.S. Pat. No.4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO 87/00195or U.S. Pat. Re. 30,985 may be used as culture media for the host cells.Any of these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleosides (such as adenosine andthymidine), antibiotics (such as Gentamycin™ drug) trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH and the like, suitably arethose previously used with the host cell selected for cloning orexpression, as the case may be, and will be apparent to the ordinaryartisan.

The host cells referred to in this disclosure encompass cells in invitro cell culture as well as cells that are within a host animal orplant.

It is further envisioned that the trk receptor of this invention may beproduced by homologous recombination, or with recombinant productionmethods utilizing control elements introduced into cells alreadycontaining DNA encoding the trk receptor.

G. Detecting Gene Amplification/Expression

Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA tThomas, Proc. Natl.Acad. Sci. USA 77, 5201-5205 (1980)!, dot blotting (DNA analysis), or insitu hybridization, using an appropriately labeled probe, based on thesequences provided herein. Various labels may be employed, most commonlyradioisotopes, particularly ³² p. However, other techniques may also beemployed, such as using biotin-modified nucleotides for introductioninto a polynucleotide. The biotin then serves as a site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed that can recognize specificduplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybridduplexes or DNA-protein duplexes. The antibodies in turn may be labeledand the assay may be carried out where the duplex is bound to thesurface, so that upon the formation of duplex on the surface, thepresence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of tissue sections andassay of cell culture or body fluids, to quantitate directly theexpression of gene product. With immunohistochemical stainingtechniques, a cell sample is prepared, typically by dehydration andfixation, followed by reaction with labeled antibodies specific for thegene product coupled, where the labels are usually visually detectable,such as enzymatic labels, fluorescent labels, luminescent labels, andthe like. A particularly sensitive staining technique suitable for usein the present invention is described by Hse et al., Am. J. Clin. Pharm.75, 734-738 (1980).

Antibodies useful for immunohistochemical staining and/or assay ofsample fluids may be either monoclonal or polyclonal, and may beprepared in any animal. Conveniently, the antibodies may be preparedagainst a native trk receptor polypeptide, or against a syntheticpeptide based on the DNA sequence provided herein as described furtherhereinbelow.

H. Purification of the trk Receptor

The trk receptor preferably is recovered from the cell culture medium asa secreted polypeptide, although it also may be recovered from host celllysates when directly expressed in a form including the membraneanchoring domain, and with or without a secretory signal.

When the trk receptor is expressed in a recombinant cell other than oneof human origin, the trk receptor is completely free of proteins orpolypeptides of human origin. However, it is necessary to purify the trkreceptor from recombinant cell proteins or polypeptides to obtainedpreparations that are substantially homogenous as to the trk receptor.As a first step, the culture medium or lysate is centrifuged to removeparticulate cell debris. The membrane and soluble protein fractions arethen separated. The trk receptor may then be purified from the solubleprotein fraction and from the membrane fraction of the culture lysate,depending on whether the trk receptor is membrane bound. The followingprocedures are exemplary of suitable purification procedures:fractionation on immunoaffinity or ion-exchange columns; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on acation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel filtration using, for example, Sephadex G-75;and protein A Sepharose columns to remove contaminants such as IgG.

Trk receptor functional derivatives in which residues have been deleted,inserted and/or substituted are recovered in the same fashion as thenative receptor chains, taking into account of any substantial changesin properties occasioned by the alteration. For example, fusion of thetrk receptor with another protein or polypeptide, e.g. a bacterial orviral antigen, facilitates purification; an immunoaffinity columncontaining antibody to the antigen can be used to absorb the fusion.Immunoaffinity columns such as a rabbit polyclonal anti-trk receptorcolumn can be employed to absorb trk receptor variant by binding to atleast one remaining immune epitope. A protease inhibitor, such as phenylmethyl sulfonyl fluoride (PMSF) also may be useful to inhibitproteolytic degradation during purification, and antibiotics may beincluded to prevent the growth of adventitious contaminants. One skilledin the art will appreciate that purification methods suitable for nativetrk receptor may require modification to account for changes in thecharacter of the trk receptor or its variants upon expression inrecombinant cell culture.

I. Covalent Modifications of trk Receptor

Covalent modifications of trk receptor are included within the scopeherein. Such modifications are traditionally introduced by reactingtargeted amino acid residues of the trk receptor with an organicderivatizing agent that is capable of reacting with selected sides orterminal residues, or by harnessing mechanisms of post-translationalmodifications that function in selected recombinant host cells. Theresultant covalent derivatives are useful in programs directed atidentifying residues important for biological activity, for immunoassaysof the trk receptor, or for the preparation of anti-trk receptorantibodies for immunoaffinity purification of the recombinant. Forexample, complete inactivation of the biological activity of the proteinafter reaction with ninhydrin would suggest that at least one arginyl orlysyl residue is critical for its activity, whereafter the individualresidues which were modified under the conditions selected areidentified by isolation of a peptide fragment containing the modifiedamino acid residue. Such modifications are within the ordinary skill inthe art and are performed without undue experimentation.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5 -imidozoyl) propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethyl-pyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to form0-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵ I or ¹³¹ I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R'--N═C═N--R') such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl, threonyl or tyrosylresidues, methylation of the α-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-861983!), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group. The molecules may further be covalentlylinked to nonproteinaceous polymers, e.g. polyethylene glycol,polypropylene glycol or polyoxyalkylenes, in the manner set forth inU.S. Ser. No. 07/275,296 or U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Derivatization with bifunctional agents is useful for preparingintramolecular aggregates of the trk receptor with polypeptides as wellas for cross-linking the trk receptor to a water insoluble supportmatrix or surface for use in assays or affinity purification. Inaddition, a study of interchain cross-links will provide directinformation on conformational structure. Commonly used cross-linkingagents include 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, homobifunctional imidoesters, andbifunctional maleimides. Derivatizing agents such as methyl-3-(p-azidophenyl) dithio! propioimidate yield photoactivatableintermediates which are capable of forming cross-links in the presenceof light. Alternatively, reactive water insoluble matrices such ascyanogen bromide activated carbohydrates and the systems reactivesubstrates described in U.S. Pat. Nos. 3,959,642; 3,969,287; 3,691,016;4,195,128; 4,247,642; 4,229,537; 4,055,635; and 4,330,440 are employedfor protein immobilization and cross-linking.

Certain post-translational modifications are the result of the action ofrecombinant host cells on the expressed polypeptide. Glutaminyl andaspariginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other post-translational modifications include hydroxylation of prolineand lysine, phosphorylation of hydroxyl groups of seryl, threonyl ortyrosyl residues, methylation of the α-amino groups of lysine, arginine,and histidine side chains T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86(1983)!.

Other derivatives comprise the novel peptides of this inventioncovalently bonded to a nonproteinaceous polymer. The nonproteinaceouspolymer ordinarily is a hydrophilic synthetic polymer, i.e. a polymernot otherwise found in nature. However, polymers which exist in natureand are produced by recombinant or in vitro methods are useful, as arepolymers which are isolated from nature. Hydrophilic polyvinyl polymersfall within the scope of this invention, e.g. polyvinylalcohol andpolyvinylpyrrolidone. Particularly useful are polyvinylalkylene etherssuch a polyethylene glycol, polypropylene glycol.

The trk receptor may be linked to various nonproteinaceous polymers,such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes,in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The trk receptor may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization, incolloidal drug delivery systems (e.g. liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th Edition, Osol, A., Ed. (1980).

J. Glycosylation Variants of the trk Receptor

The native trk receptors are glycoproteins. Variants having aglycoslation pattern which differs from that of any native amino acidsequence which might be present in the molecules of the presentinvention are within the scope herein. For ease, changes in theglycosylation pattern of a native polypeptide are usually made at theDNA level, essentially using the techniques discussed hereinabove withrespect to the amino acid sequence variants.

Chemical or enzymatic coupling of glycosydes to the trk receptor of themolecules of the present invention may also be used to modify orincrease the number or profile of carbohydrate substituents. Theseprocedures are advantageous in that they do not require production ofthe polypeptide that is capable of O-linked (or N-linked) glycosylation.Depending on the coupling mode used, the sugar(s) may be attached to (a)arginine and histidine, (b) free carboxyl groups, (c) free hydroxylgroups such as those of cysteine, (d) free sulfhydryl groups such asthose of serine, threonine, or hydroxyproline, (e) aromatic residuessuch as those of phenylalanine, tyrosine, or tryptophan or (f) the amidegroup of glutamine. These methods are described in WO 87/05330(published Sep. 11, 1987), and in Aplin and Wriston, CRC Crit. Rev.Biochem., pp. 259-306.

Carbohydrate moieties present on a polypeptide may also be removedchemically or enzymatically. Chemical deglycosylation requires exposureto trifluoromethanesulfonic acid or an equivalent compound. Thistreatment results in the cleavage of most or all sugars, except thelinking sugar, while leaving the polypeptide intact. Chemicaldeglycosylation is described by Hakimuddin et al., Arch. Biochem.Biophys. 259, 52 (1987) and by Edge et al., Anal. Biochem. 118, 131(1981). Carbohydrate moieties can be removed by a variety of endo- andexoglycosidases as described by Thotakura et al., Meth. Enzymol. 138,350 (1987). Glycosylation is suppressed by tunicamycin as described byDuskin et al., J. Biol. Chem. 257, 3105 (1982). Tunicamycin blocks theformation of protein-N-glycosydase linkages.

Glycosylation variants can also be produced by selecting appropriatehost cells of recombinant production. Yeast, for example, introduceglycosylation which varies significantly from that of mammalian systems.Similarly, mammalian cells having a different species (e.g. hamster,murine, insect, porcine, bovine or ovine) or tissue (e.g. lung, liver,lymphoid, mesenchymal or epidermal) origin than the source of the nativetrk receptor, are routinely screened for the ability to introducevariant glycosylation.

K. trk Receptor-Immunoglobulin Chimeras (Immunoadhesins)

Immunoadhesins are chimeric antibody-like molecules that combine thefunctional domain(s) of a binding protein (usually a receptor, acell-adhesion molecule or a ligand) with the an immunoglobulin sequence.The immunoglobulin sequence preferably (but not necessarily) is animmunoglobulin constant domain.

Immunoglobulins (Ig) and certain variants thereof are known and manyhave been prepared in recombinant cell culture. For example, see U.S.Pat. Nos. 4,745,055; EP 256,654; Faulkner et al., Nature 2:286 (1982);EP 120,694; EP 125,023; Morrison, J. Imun. 123:793 (1979); Kohler etal., Proc. Nat'l. Acad. Sci. USA 77:2197 (1980); Raso et al., CancerRes. 41:2073 (1981); Morrison et al., Ann. Rev. Immunol. 2:239 (1984);Morrison, Science 229:1202 (1985); Morrison et al., Proc. Nat'l. Acad,Sci. USA 81:6851 (1984); EP 255,694; EP 266,663; and WO 88/03559.Reassorted immunoglobulin chains also are known. See for example U.S.Pat. No. 4,444,878; WO 88/03565; and EP 68,763 and references citedtherein. The immunoglobulin moiety in the chimeras of the presentinvention may be obtained from IgG-1, IgG-2, IgG-3 or IgG-4 subtypes,IgA, IgE, IgD or IgM, but preferably IgG-1 or IgG-3.

Chimeras constructed from a receptor sequence linked to an appropriateimmunoglobulin constant domain sequence (immunoadhesins) are known inthe art. Immunoadhesins reported in the literature include fusions ofthe T cell receptor^(*) Gascoigne et al., Proc. Natl. Acad. Sci. USA 84,2936-2940 (1987)!; CD4^(*) Capon et al., Nature 337, 525-531 (1989);Traunecker et al., Nature 339, 68-70 (1989); Zettmeissl et al., DNA CellBiol. USA 9, 347-353 (1990); Byrn et al., Nature 344, 667-670 (1990)!;L-selectin (homing receptor) Watson et al., J. Cell. Biol. 110,2221-2229 (1990); Watson et al., Nature 349, 164-167 (1991)!; CD44^(*)Aruffo et al., Cell 61, 1303-1313 (1990)!; CD28^(*) and B7^(*) Linsleyet al., J. Exp. Med. 173, 721-730 (1991); CTLA-4^(*) Lisley et al., J.Exp. Med. 174, 561-569 (1991)!; CD22^(*) Stamenkovic et al., Cell 66.1133-1144 (1991)!; TNF receptor Ashkenazi et al., Proc. Natl. Acad. Sci.USA 88, 10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27,2883-2886 (1991); Peppel et al., J. Exp. Med. 174, 1483-1489 (1991)!; NPreceptors Bennett et al., J. Biol. Chem. 266, 23060-23067 (1991)!; IgEreceptor α^(*) Ridgway and Gorman, J. Cell. Biol. 115, abstr. 1448(1991)!; HGF receptor Mark, M. R. et al., 1992, J. Biol. Chem.submitted!, where the asterisk (*) indicates that the receptor is memberof the immunoglobulin superfamily.

The simplest and most straightforward immunoadhesin design combined thebinding region(s) of the `adhesin` protein with the hinge and Fc regionsof an immunoglobulin heavy chain. Ordinarily, when preparing the trkreceptor-immunoglobulin chimeras of the present invention, nucleic acidencoding the extracellular domain or a fragment thereof of a desired trkreceptor will be fused C-terminally to nucleic acid encoding theN-terminus of an immunoglobulin constant domain sequence, howeverN-terminal fusions are also possible.

Typically, in such fusions the encoded chimeric polypeptide will retainat least functionally active hinge, CH2 and CH3 domains of the constantregion of an immunoglobulin heavy chain. Fusions are also made to theC-terminus of the Fc portion of a constant domain, or immediatelyN-terminal to the CH1 of the heavy chain or the corresponding region ofthe light chain.

The precise site at which the fusion is made is not critical; particularsites are well known and may be selected in order to optimize thebiological activity, secretion or binding characteristics of the trkreceptor-immunoglobulin chimeras.

In some embodiments, the trk receptor-immunoglobulin chimeras areassembled as monomers, or hetero- or homo-multimers, and particularly asdimers or tetramers, essentially as illustrated in WO 91/08298.

In a preferred embodiment, the trk receptor extracellular domainsequence, which preferably includes the second immunoglobulin-likedomain, is fused to the N-terminus of the C-terminal portion of anantibody (in particular the Fc domain), containing the effectorfunctions of an immunoglobulin, e.g. immunoglobulin G₁ (IgG-1). It ispossible to fuse the entire heavy chain constant region to the trkreceptor extracellular domain sequence. However, more preferably, asequence beginning in the hinge region just upstream of the papaincleavage site (which defines IgG Fc chemically; residue 216, taking thefirst residue of heavy chain constant region to be 114 Kobet et al.,supra!, or analogous sites of other immunoglobulins) is used in thefusion. In a particularly preferred embodiment, the trk receptor aminoacid sequence is fused to the hinge region and CH2 and CH3 or CH1,hinge, CH2 and CH3 domains of an IgG-1, IgG-2, or IgG-3 heavy chain. Theprecise site at which the fusion is made is not critical, and theoptimal site can be determined by routine experimentation.

In some embodiments, the trk receptor-immunoglobulin chimeras areassembled as multimers, and particularly as homo-dimers or -tetramers.Generally, these assembled immunoglobulins will have known unitstructures. A basic four chain structural unit is the form in which IgG,IgD, and IgE exist. A four unit is repeated in the higher molecularweight immunoglobulins; IgM generally exists as a pentamer of basic fourunits held together by disulfide bonds. IgA globulin, and occasionallyIgG globulin, may also exist in multimeric form in serum. In the case ofmultimer, each four unit may be the same or different.

Various exemplary assembled trk receptor-immunoglobulin chimeras withinthe scope herein are schematically diagrammed below:

(a) AC_(L) -AC_(L) ;

(b) AC_(H) - AC_(H), AC_(L) -AC_(H), AC_(L) -V_(H) C_(H), or V_(L) C_(L)-AC_(H) !;

(c) AC_(L) -AC_(H) - AC_(L) -AC_(H), AC_(L) -V_(H) C_(H), V_(L) C_(L)-AC_(H), or V_(L) C_(L) -V_(H) C_(H) !;

(d) AC_(L) -V_(H) C_(H) - AC_(H), or AC_(L) -V_(H) C_(H), or V_(L) C_(L)-AC_(H) !;

(e) V_(L) C_(L) -AC_(H) - AC_(L) -V_(H) C_(H), or V_(L) C_(L) -AC_(H) !;and

(f) A-Y!_(n) - V_(L) C_(L) -V_(H) C_(H) !₂,

wherein

each A represents identical or different trk receptor amino acidsequences;

V_(L) is an immunoglobulin light chain variable domain;

V_(H) is an immunoglobulin heavy chain variable domain;

C_(L) is an immunoglobulin light chain constant domain;

C_(H) is an immunoglobulin heavy chain constant domain;

n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent.

In the interests of brevity, the foregoing structures only show keyfeatures; they do not indicate joining (J) or other domains of theimmunoglobulins, nor are disulfide bonds shown. However, where suchdomains are required for binding activity, they shall be constructed asbeing present in the ordinary locations which they occupy in theimmunoglobulin molecules.

Alternatively, the trk receptor extracellular domain sequences can beinserted between immunoglobulin heavy chain and light chain sequencessuch that an immunoglobulin comprising a chimeric heavy chain isobtained. In this embodiment, the trk receptor sequences are fused tothe 3' end of an immunoglobulin heavy chain in each arm of animmunoglobulin, either between the hinge and the CH2 domain, or betweenthe CH2 and CH3 domains. Similar constructs have been reported byHoogenboom, H. R. et al., Mol. Immunol. 28, 1027-1037 (1991).

Although the presence of an immunoglobulin light chain is not requiredin the immunoadhesins of the present invention, an immunoglobulin lightchain might be present either covalently associated to an trkreceptor-immunoglobulin heavy chain fusion polypeptide, or directlyfused to the trk receptor extracellular domain. In the former case, DNAencoding an immunoglobulin light chain is typically coexpressed with theDNA encoding the trk receptor-immunoglobulin heavy chain fusion protein.Upon secretion, the hybrid heavy chain and the light chain will becovalently associated to provide an immunoglobulin-like structurecomprising two disulfide-linked immunoglobulin heavy chain-light chainpairs. Method suitable for the preparation of such structures are, forexample, disclosed in U.S. Pat. No. 4,816,567 issued Mar. 28, 1989.

In a preferred embodiment, the immunoglobulin sequences used in theconstruction of the immunoadhesins of the present invention are from anIgG immunoglobulin heavy chain constant domain. For humanimmunoadhesins, the use of human IgG1 and IgG3 immunoglobulin sequencesis preferred. A major advantage of using IgG1 is that IgG1immunoadhesins can be purified efficiently on immobilized protein A. Incontrast, purification of IgG3 requires protein G, a significantly lessversatile medium. However, other structural and functional properties ofimmunoglobulins should be considered when choosing the Ig fusion partnerfor a particular immunoadhesin construction. For example, the IgG3 hingeis longer and more flexible, so it can accommodate larger `adhesin`domains that may not fold or function properly when fused to IgG1.Another consideration may be valency; IgG immunoadhesins are bivalenthomodimers, whereas Ig subtypes like IgA and IgM may give rise todimeric or pentameric structures, respectively, of the basic Ighomodimer unit. For trk-Ig immunoadhesins designed for in vivoapplication, the pharmacokinetic properties and the effector functionsspecified by the Fc region are important as well. Although IgG1, IgG2and IgG4 all have in vivo half-lives of 21 days, their relativepotencies at activating the complement system are different. IgG4 doesnot activate complement, and IgG2 is significantly weaker at complementactivation than IgG1. Moreover, unlike IgG1, IgG2 does not bind to Fcreceptors on mononuclear cells or neutrophils. While IgG3 is optimal forcomplement activation, its in vivo half-life i approximately one thirdof the other IgG isotypes. Another important consideration forimmunoadhesins designed to be used as human therapeutics is the numberof allotypic variants of the particular isotype. In general, IgGisotypes with fewer serologically-defined allotypes are preferred. Forexample, IgG1 has only four serologically-defined allotypic sites, twoof which (G1m and 2) are located in the Pc region; and one of thesesites G1m1, is non-immunogenic. In contrast, there are 12serologically-defined allotypes in IgG3, all of which are in the Fcregion; only three of these sites (G3m5, 11 and 21) have one allotypewhich is nonimmunogenic. Thus, the potential immunogenicity of a γ3innunoadhesin is greater than that of a γ1 immunoadhesin.

In designing the trk-Ig immunoadhesins of the present invention domainthat are not required for neurotrophin binding and/or biologicalactivity may be deleted. In such structures, it is important to placethe fusion junction at residues that are located between domains, toavoid misfolding. With respect to the parental immunoglobulin, a usefuljoining point is just upstream of the cysteines of the hinge that formthe disulfide bonds between the two heavy chains. In a frequently useddesign, the codon for the C-terminal residue of the `adhesin` (trk) partof the molecule is placed directly upstream of the codons for thesequence DKTHTCPPCP of the IgG1 hinge region.

The general methods suitable for the construction and expression ofimmunoadhesins are the same those disclosed hereinabove with regard to(native or variant) trk receptors. trk-Ig immunoadhesins are mostconveniently constructed by fusing the cDNA sequence encoding the trkportion in-frame to an Ig cDNA sequence. However, fusion to genomic Igfragments can also be used see, e.g. Gascoigne et al., Proc. Natl. Acad.Sci. USA 84, 2936-2940 (1987); Aruffo et al., Cell 61, 1303-1313 (1990);Stamenkovic et al., Cell 66, 1133-1144 (1991)!. The latter type offusion requires the presence of Ig regulatory sequences for expression.cDNAs encoding IgG heavy-chain constant regions can be isolated based onpublished sequence from cDNA libraries derived from spleen or peripheralblood lymphocytes, by hybridization or by polymerase chain reaction(PCR) techniques. The cDNAs encoding the `adhesin` and the Ig parts ofthe immunoadhesin are inserted in tandem into a plasmid vector thatdirects efficient expression in the chosen host cells. For expression inmammalian cells pRK5-based vectors Schall et al., Cell 61, 361-370(1990)! and CDM8-based vectors Seed, Nature 329, 840 (1989)!. The exactjunction can be created by removing the extra sequences between thedesigned junction codons using oligonucleotide-directed deletionalmutagenesis Zoller and Smith, Nucleic Acids Res. 10, 6487 (1982); Caponet al., Nature 337, 525-531 (1989)!. Synthetic oligonucleotides can beused, in which each half is complementary to the sequence on either sideof the desired junction; ideally, these are 36 to 48-mers.Alternatively, PCR techniques can be used to join the two parts of themolecule in-frame with an appropriate vector.

The choice of host cell line for the expression of trk-Ig immunoadhesinsdepends mainly on the expression vector. Another consideration is theamount of protein that is required. Milligram quantities often can beproduced by transient transfections. For example, the adenovirusEIA-transformed 293 human embryonic kidney cell line can be transfectedtransiently with pRK5-based vectors by a modification of the calciumphosphate method to allow efficient immunoadhesin expression. CDMB-basedvectors can be used to transfect COS cells by the DEAE-dextran method(Aruffo et al., Cell 61, 1303-1313 (1990); Zettmeissl et al., DNA CellBiol. (US) 9, 347-353 (1990)!. If larger amounts of protein are desired,the immunoadhesin can be expressed after stable transfection of a hostcell line. For example, a pRK5-based vector can be introduced intoChinese hamster ovary (CHO) cells in the presence of an additionalplasmid encoding dihydrofolate reductase (DHFR) and conferringresistance to G418. Clones resistant to G418 can be selected in culture;these clones are grown in the presence of increasing levels of DHFRinhibitor methotrexate; clones are selected, in which the number of genecopies encoding the DHFR and immunoadhesin sequences is co-amplified. Ifthe immunoadhesin contains a hydrophobic leader sequence at itsN-terminus, it is likely to be processed and secreted by the transfectedcells. The expression of immunoadhesins with more complex structures mayrequire uniquely suited host cells; for example, components such aslight chain or J chain may be provided by certain myeloma or hybridomacell hosts Gascoigne et al., 1987, supra; Martin et al., J. Virol. 67,3561-3568 (1993)!.

Immunoadhesins can be conveniently purified by affinity chromatography.The suitability of protein A as an affinity ligand depends on thespecies and isotype of the immunoglobulin Fc domain that is used in thechimera. Protein A can be used to purify immunoadhesins that are basedon human γ1, γ2, or γ4 heavy chains Lindmark et al., J. Immunol. Meth.62, 1-13 (1983)!. Protein G is recommended for all mouse isotypes andfor human γ3 Guss et al., EMBO J. 5, 15671575 (1986)!. The matrix towhich the affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Theconditions for binding an immunoadhesin to the protein A or G affinitycolumn are dictated entirely by the characteristics of the Fc domain;that is, its species and isotype. Generally, when the proper ligand ischosen, efficient binding occurs directly from unconditioned culturefluid. One distinguishing feature of immunoadhesins is that, for humanγ1 molecules, the binding capacity for protein A is somewhat diminishedrelative to an antibody of the same Fc type. Bound immunoadhesin can beefficiently eluted either at acidic pH (at or above 3.0), or in aneutral pH buffer containing a mildly chaotropic salt. This affinitychromatography step can result in an immunoadhesin preparation thatis >95% pure.

Other methods known in the art can be used in place of, or in additionto, affinity chromatography on protein A or G to purify immunoadhesins.Immunoadhesins behave similarly to antibodies in thiophilic gelchromatography Hutchens and Porath, Anal. Biochem. 159, 217-226 (1986)!and immobilized metal chelate chromatography Al-Mashikhi and Makai, J.Dairy Sci. 71, 1756-1763 (1988)!. In contrast to antibodies, however,their behavior on ion exchange columns is dictated not only by theirisoelectric points, but also by a charge dipole that may exist in themolecules due to their chimeric nature.

If desired, the immunoadhesins can be made bispecific, that is, directedagainst two distinct ligands. Thus, the immunoadhesins of the presentinvention may have binding specificities for two distinct neurotrophins,or may specifically bind to a neurotrophin and to an other determinantspecifically expressed on the cells expressing the neurotrophin to whichthe trk portion of the immunoadhesin structure binds. For bispecificmolecules, trimeric molecules, composed of a chimeric antibody heavychain in one arm and a chimeric antibody heavy chain-light chain pair inthe other arm of their antibody-like structure are advantageous, due toease of purification. In contrast to antibody-producing quadromastraditionally used for the production of bispecific immunoadhesins,which produce a mixture of ten tetramers, cells transfected with nucleicacid encoding the three chains of a trimeric immunoadhesin structureproduce a mixture of only three molecules, and purification of thedesired product from this mixture is correspondingly easier.

L. trk Receptor Antibody Preparation

(i) Polyclonal Antibodies

Polyclonal antibodies to the trk receptor generally are raised inanimals by multiple subcutaneous (sc) or intraperitoneal (ip) injectionsof the trk receptor and an adjuvant. It may be useful to conjugate thetrk receptor or a fragment containing the target amino acid sequence toa protein that is immunogenic in the species to be immunized, e.g.keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example maleimidobenzoyl sulfosuccinimide ester (conjugation throughcysteine residues), N-hydroxysuccinimide (through lysine residues),glytaraldehyde, succinic anhydride, SOCl₂, or R¹ N═C═NR, where R and R¹are different alkyl groups.

Animals are immunized against the immunogenic conjugates or derivativesby combining 1 mg of 1 μg of conjugate (for rabbits or mice,respectively) with 3 volumes of Freud's complete adjuvant and injectingthe solution intradermally at multiple sites. One month later theanimals are boosted with 1/5 to 1/10 the original amount of conjugate inFreud's complete adjuvant by subcutaneous injection at multiple sites. 7to 14 days later the animals are bled and the serum is assayed foranti-trk receptor antibody titer. Animals are boosted until the titerplateaus. Preferably, the animal boosted with the conjugate of the sametrk receptor, but conjugated to a different protein and/or through adifferent cross-linking reagent. Conjugates also can be made inrecombinant cell culture as protein fusions. Also, aggregating agentssuch as alum are used to enhance the immune response.

(ii) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Thus, the modifier"monoclonal" indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the anti-trk receptor monoclonal antibodies of theinvention may be made using the hybridoma method first described byKohler & Milstein, Nature 256:495 (1975), or may be made by recombinantDNA methods Cabilly, et al., U.S. Pat. No. 4,816,567!.

In the hybridoma method, a mouse or other appropriate host animal, suchas hamster is immunized as hereinabove described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the protein used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell Goding, Monoclonal Antibodies:Principles and Practice, pp.59-103 (Academic Press, 1986)!.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh level expression of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against trk receptor.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson & Pollard, Anal. Biochem.107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.Goding, Monoclonal Antibodies: Principles and Practice, pp.59-104(Academic Press, 1986). Suitable culture media for this purpose include,for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies of the invention is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences,Morrison, et al., Proc. Nat. Acad. Sci. 81, 6851 (1984), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. In thatmanner, "chimeric" or "hybrid" antibodies are prepared that have thebinding specificity of an anti-trk monoclonal antibody herein.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody of the invention, or they aresubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for an trkreceptor and another antigen-combining site having specificity for adifferent antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

For diagnostic applications, the antibodies of the invention typicallywill be labeled with a detectable moiety. The detectable moiety can beany one which is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as ³ H, ¹⁴ C, ³² P, ³⁵ S, or ¹²⁵ I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; biotin; radioactive isotopic labels, such as,e.g., ¹²⁵ I, ³² P, ¹⁴ C, or ³ H, or an enzyme, such as alkalinephosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); andNygren, J. Histochem. and Cytochem. 30:407 (1982).

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).

Competitive binding assays rely on the ability of a labeled standard(which may be an trk receptor or an immunologically reactive portionthereof) to compete with the test sample analyte (trk receptor) forbinding with a limited amount of antibody. The amount of trk receptor inthe test sample is inversely proportional to the amount of standard thatbecomes bound to the antibodies. To facilitate determining the amount ofstandard that becomes bound, the antibodies generally are insolubilizedbefore or after the competition, so that the standard and analyte thatare bound to the antibodies may conveniently be separated from thestandard and analyte which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, of the proteinto be detected. In a sandwich assay, the test sample analyte is bound bya first antibody which is immobilized on a solid support, and thereaftera second antibody binds to the analyte, thus forming an insoluble threepart complex. David & Greene, U.S. Pat No. 4,376,110. The secondantibody may itself be labeled with a detectable moiety (direct sandwichassays) or may be measured using an anti-immunoglobulin antibody that islabeled with a detectable moiety (indirect sandwich assay). For example,one type of sandwich assay is an ELISA assay, in which case thedetectable moiety is an enzyme.

(iii) Humanized Antibodies

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as "import" residues, whichare typically taken from an "import" variable domain. Humanization canbe essentially performed following the method of Winter and co-workersJones et al., Nature 321, 522-525 (1986); Riechmann et al., Nature 332,323-327 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988)!, bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such "humanized" antibodiesare chimeric antibodies (Cabilly, supra), wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three dimensional models ofthe parental and humanized sequences. Three dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e. the analysis of residues thatinfluence the ability of the candidate immuoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from theconsensus and import sequence so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. For furtherdetails see U.S. application Ser. No. 07/934,373 filed 21 August 192,which is a continuation-in-part of application Ser. No. 07/715,272 filedJun. 14, 1991.

(iv) Human Antibodies

Human monoclonal antibodies can be made by the hybridoma method. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described, for example, by Kozbor,J. Immunol. 133, 3001 (1984), and Brodeur, et al., Monoclonal AntibodyProduction Techniques and Applications, pp.51-63 (Marcel Dekker, Inc.,New York, 1987).

It is now possible to produce transgenic animals (e.g. mice) that arecapable, upon immunization, of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g. Jakobovits et al.,Proc. Natl. Acad. Sci. USA 90, 2551-255 (1993); Jakobovits et al.,Nature 362, 255-258 (1993).

Alternatively, the phage display technology (McCafferty et al., Nature348, 552-553 1990!) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimicks someof the properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g. Johnson, Kevin S. andChiswell, David J., Current Opinion in Structural Biology 3, 564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature 352, 624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222, 581-597 (1991), or Griffith et al., EMBO J.12, 725-734 (1993). In a natural immune response, antibody genesaccumulate mutations at a high rate (somatic hypermutation). Some of thechanges introduced will confer higher affinity, and B cells displayinghigh-affinity surface immunoglobulin are preferentially replicated anddifferentiated during subsequent antigen challenge. This natural processcan be mimicked by employing the technique known as "chain shuffling"(Marks et al., Bio/Technol. 10, 779-783 1992!). In this method, theaffinity of "primary" human antibodies obtained by phage display can beimproved by sequentially replacing the heavy and light chain V regiongenes with repertoires of naturally occurring variants (repertoires) ofV domain genes obtained from unimmunized donors. This techniques allowsthe production of antibodies and antibody fragments with affinities inthe nM range. A strategy for making very large phage antibodyrepertoires (also known as "the mother-of-all libraries") has beendescribed by Waterhouse et al., Nucl. Acids Res. 21, 2265-2266 (1993),and the isolation of a high affinity human antibody directly from suchlarge phage library is reported by Griffith et al., EMBO J. (1994), inpress. Gene shuffling can also be used to derive human antibodies fromrodent antibodies, where the human antibody has similar affinities andspecificities to the starting rodent antibody. According to this method,which is also referred to as "epitope imprinting", the heavy or lightchain V domain gene of rodent antibodies obtained by is phage displaytechnique is replaced with a repertoire of human V domain genes,creating rodent-human chimeras. Selection on antigen results inisolation of human variable capable of restoring a functionalantigen-binding site, i.e. the epitope governs (imprints) the choice ofpartner. When the process is repeated in order to replace the remainingrodent V domain, a human antibody is obtained (see PCT patentapplication WO 93/06213, published Apr. 1, 1993). Unlike traditionalhumanization of rodent antibodies by CDR grafting, this techniqueprovides completely human antibodies, which have no framework or CDRresidues of rodent origin.

(v) Bispecific Antibodies

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is for atrk receptor, the other one is for any other antigen, and preferably foranother receptor or receptor subunit. For example, bispecific antibodiesspecifically binding a trk receptor and neurotrophic factor, or twodifferent trk receptors are within the scope of the present invention.

Methods for making bispecific antibodies are known in the art.

Traditionally, the recombinant production of bispecific antibodies isbased on the coexpression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities(Millstein and Cuello, Nature 305, 537-539 (1983)). Because of therandom assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of 10 differentantibody molecules, of which only one has the correct bispecificstructure. The purification of the correct molecule, which is usuallydone by affinity chromatography steps, is rather cumbersome, and theproduct yields are low. Similar procedures are disclosed in PCTapplication publication No. WO 93/08829 (published May 13, 1993), and inTraunecker et al., EMBO 10, 3655-3659 (1991).

According to a different and more preferred approach, antibody variabledomains with the desired binding specificities (antibody-antigencombining sites) are fused to immunoglobulin constant domain sequences.The fusion preferably is with an immunoglobulin heavy chain constantdomain, comprising at least part of the hinge, CH2 and CH3 regions. Itis preferred to have the first heavy chain constant region (CH1)containing the site necessary for light chain binding, present in atleast one of the fusions. DNAs encoding the immunoglobulin heavy chainfusions and, if desired, the immunoglobulin light chain, are insertedinto separate expression vectors, and are cotransfected into a suitablehost organism. This provides for great flexibility in adjusting themutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance. In a preferred embodiment of this approach,the bispecific antibodies are composed of a hybrid immunoglobulin heavychain with a first binding specificity in one arm, and a hybridimmunoglobulin heavy chain-light chain pair (providing a second bindingspecificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation. This approach is disclosed incopending application Ser. No. 07/931,811 filed Aug. 17, 1992.

For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology 121, 210 (1986).

(v) Heteroconjugate Antibodies

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for treatment of HIV infection (PCT application publication Nos. WO91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

M. Use of the trk-Ig Immunoadhesins

(i) Ligand Binding

As in antibodies, the Fc region of immunoadhesins provides a convenienthandle not only for purification, but also for capture and detection.This is useful for quantitation of the immunoadhesin (e.g., intransfected cell supernatants) using a sandwich ELISA with two differentanti-Fc antibodies. In addition, the Fc handle facilitates investigatingthe interaction of the trk portion with the correspondingneurotrophin(s). For example, a microtiter plate binding assay formatcan be used, in which the immunoadhesin is immobilized onto wells thathave been pre-coated with anti-Fc antibody. This positions theimmunoadhesin in an orientation which leaves the trk portion accessiblefor binding by a cognate neurotrophin ligand. The ligand is then addedand incubated with the immobilized immunoadhesin. After removal of theunbound ligand by washing, binding is quantitated by countingradioactivity if the neurotrophin ligand is radiolabeled, or byanti-neurotrophin antibodies. Nonspecific binding can be determined byomitting the immunoadhesin or by including an isotype-matchedimmunoadhesin with an irrelevant `adhesin` portion. This assay formatcan be used for the diagnosis of pathological conditions characterizedby the under- or overexpression of certain neurotrophins, and is alsouseful in comparing the binding of various neurotrophic factors to atrkA, trkB or trkC receptor, and in efforts aimed at finding new ligandsfor trk receptors, e.g., in screening libraries of synthetic or naturalorganic compounds.

(ii) Ligand Identification/isolation

Another area in which trk-Ig innunoadhesins can be used is search forfurther neurotrophins in the human or in various animal species, and forpurifying such ligands. Ligands identified so far by this approachinclude two L-selectin ligands, GlyCAM-1 and CD34, which were identifiedand purified using an L-selectin-IgG affinity column (Imai et al., J.Cell. Biol. 113, 1213-1221 (1991); Watson et al., J. Cell. Biol, 110,2221-2229 (1990); Watson et al., J. Cell. Biol. 349, 164-167 (1991)!.

(iii) Production of Large Quantities of Purified Soluble trk receptors

The structural similarity between immoadhesins and antibodies suggestedthat it might be possible to cleave immunoadhesins by proteolyticenzymes such as papain, to generate Fd-like fragments containing the`adhesin` portion. In order to provide a more generic approach forcleavage of immunoadhesins, proteases which are highly specific fortheir target sequence are to be used. A protease suitable for thispurpose is an engineered mutant of subtilisin BPN, which recognizes andcleaves the sequence AAHYTL. Introduction of this target sequence intothe support hinge region of a trk-IgG1 immunoadhesin facilitates highlyspecific cleavage between the Fc and trk domains. The immunoadhesin ispurified by protein A chromatography and cleaved with an immobilized forof the enzyme. Cleavage results in two products; the Fc region and thetrk region. These fragments can be separated easily by a second passageover a protein A column to retain the Fc and obtain the purified trkfragments in the flow-through fractions. A similar approach can be usedto generate a dimeric trk portion, by placing the cleavable sequence atthe lower hinge.

N. Use of trk Receptors

(i) Kinase Receptor Activation Assay

The trk receptors can be used in the kinase receptor activation (KIRA)assay. This ELISA-type assay is suitable for qualitative or quantitativemeasurement of kinase activation by measuring the autophosphorylation ofthe kinase domain of a receptor protein tyrosine kinase (rPTK, e.g. trkreceptor), as well as for identification and characterization ofpotential agonist or antagonists of a selected rPTK. The first stage ofthe assay involves phosphorylation of the kinase domain of a kinasereceptor, e.g. a trk receptor, wherein the receptor is present in thecell membrane of a eukaryotic cell. The receptor may be an endogenousreceptor or nucleic acid encoding the receptor, or a receptor construct,may be transformed into the cell. Typically, a first solid phase (e.g.,a well of a first assay plate) is coated with a substantiallyhomogeneous population of such cells (usually a mammalian cell line) sothat the cells adhere to the solid phase. Often, the cells are adherentand thereby adhere naturally to the first solid phase. If a "receptorcontruct" is used, it usually comprises a fusion of a kinase receptorand a flag polypeptide. The flag polypeptide is recognized by thecapture agent, often a capture antibody, in the ELISA part of the assay.An analyte is then added to the wells having the adhering cells, suchthat the tyrosine kinase receptor (e.g. trk receptor) is exposed to (orcontacted with) the analyte. This assay enables identification ofagonist and antagonist ligands for the tyrosine kinase receptor ofinterest (e.g. trk A, trk B or trk C). In order to detect the presenceof an antagonist ligand which blocks binding of an agonist to thereceptor, the adhering cells are exposed to the suspected antagonistligand first, and then to the agonist ligand, so that competitiveinhibition of receptor binding and activation can be measured. Also, theassay can identify an antagonist which binds to the agonist ligand andthereby reduces or eliminates its ability to bind to, and activate, therPTK. To detect such an antagonist, the suspected antagonist and theagonist for the rPTK are incubated together and the adhering cells arethen exposed to this mixture of ligands. Following exposure to theanalyte, the adhering cells are solubilized using a lysis buffer (whichhas a solubilizing detergent therein) and gentle agitation, therebyreleasing cell lysate which can be subjected to the ELISA part of theassay directly, without the need for concentration or clarification ofthe cell lysate. The cell lysate thus prepared is then ready to besubjected to the ELISA stage of the assay. As a first step in the ELISAstage, a second solid phase (usually a well of an ELISA microtiterplate) is coated with a capture agent (often a capture antibody) whichbinds specifically to the tyrosine kinase receptor, or, in the case of areceptor construct, to the flag polypeptide. Coating of the second solidphase is carried out so that the capture agent adheres to the secondsolid phase. The capture agent is generally a monoclonal antibody, but,as is described in the examples herein, polyclonal antibodies may alsobe used. The cell lysate obtained is then exposed to, or contacted with,the adhering capture agent so that the receptor or receptor constructadheres to (or is captured in) the second solid phase. A washing step isthen carried out, so as to remove unbound cell lysate, leaving thecaptured receptor or receptor construct. The adhering or capturedreceptor or receptor construct is then exposed to, or contacted with, ananti-phosphotyrosine antibody which identifies phosphorylated tyrosineresidues in the tyrosine kinase receptor. In the preferred embodiment,the anti-phosphotyrosine antibody is conjugated (directly or indirectly)to an enzyme which catalyses a color change of a non-radioactive colorreagent. Accordingly, phosphorylation of the receptor can be measured bya subsequent color change of the reagent. The enzyme can be bound to theanti-phosphotyrosine antibody directly, or a conjugating molecule (e.g.,biotin) can be conjugated to the anti-phosphotyrosine antibody and theenzyme can be subsequently bound to the anti-phosphotyrosine antibodyvia the conjugating molecule. Finally, binding of theanti-phosphotyrosine antibody to the captured receptor or receptorconstruct is measured, e.g., by a color change in the color reagent.

(ii) Therapeutic Use

The trkB and trkC receptor polypeptides of the present invention as wellas the antibodies specifically binding such receptors, either inmonospecific or bispecific or heteroconjugate form, are useful insignaling, enhancing or blocking the biological activity ofneurotrophins capable of binding at least one of these receptors. Thetrk-Ig immunoadhesins of the present invention have been found to blockthe interaction of the trk receptors with their neurotrophic ligands,and thereby inhibit neurotrophin biological activity. This antagonistactivity is believed to be useful in the treatment of pathologicalconditions associated with endogenous neurotrophin production, such asinflammatory pain (trkA-immunoadhesin; see Example 5), pancreas(trkB-immunoadhesin), kidney disorders, lung disorders, cardiovasculardisorders (trkC-immunoadhesins), various types of tumors (trkA-, trkB-and trkC-immunoadhesins), aberrant sprouting in epilepsy, psychiatricdisorders (trkB- and trkC-immunoadhesins). Human immunoadhesins can bebased on human sequences of both the trk and Ig portions of themolecule, such that the only novel sequence which may be recognized as`foreign` by the human immune system is the junction. Therefore, humanimmunoadhesins, in contrast to chimeric (humanized) antibodies, areminimally immunogenic in humans. This reduced immunogenicity is animportant advantage especially for indications that require multipleadministrations.

Therapeutic formulations of the present invention are prepared forstorage by mixing the active ingredient having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid; low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, Pluronics or PEG.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

The formulations to be used for In vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.

Therapeutic compositions herein generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

Suitable examples of sustained release preparations includesemipermeable polymer matrices in the form of shaped articles, e.g.films, or microcapsules. Sustained release matrices include polyesters,hydrogels, polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymersof L-glutamic acid and gamma ethyl-L-glutamate (U. Sidman et al., 1983,"Biopolymers" 22 (1): 547-556), poly (2-hydroxyethyl-methacrylate) (R.Langer, et al., 1981, "J. Biomed. Mater. Res." 15: 167-277 and R.Langer, 1982, "Chem. Tech." 12: 98-105), ethylene vinyl acetate (R.Langer et al., Id.) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988A).Sustained release compositions also include liposomes. Liposomescontaining a molecule within the scope of the present invention areprepared by methods known per se: DE 3,218,121A; Epstein et al., 1985,"Proc. Natl. Acad. Sci. USA" 82: 3688-3692; Hwang et al., 1980, "Proc.Natl. Acad. Sci. USA" 77: 4030-4034; EP 52322A; EP 36676A; EP 88046A; EP143949A; EP 142641A; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324A. Ordinarily the liposomesare of the small (about 200-800 Angstroms) unilamelar type in which thelipid content is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal NT-4 therapy.

An effective amount of a molecule of the present invention to beemployed therapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. A typical daily dosage might range fromabout 1 μg/kg to up to 100 mg/kg or more, depending on the factorsmentioned above. Typically, the clinician will administer a molecule ofthe present invention until a dosage is reached that provides therequired biological effect. The progress of this therapy is easilymonitored by conventional assays.

The invention will be further illustrated by the following non-limitingexamples. For the experiments described in the Examples, human braincDNA, poly α+ RNA, genomic and cDNA libraries were obtained fromClontech (Palo Alto, Calif.). pGEM was obtained from Promega (Madison,Wis.), restriction enzymes from New England Biolabs (Beverly, Mass.).Taq polymerase was from Perkin-Elmer (Norwalk, Conn.), while all otherenzymes, frozen competent E. coli and tissue culture media werepurchased from Gibco-BRL (Raithersburg, Md.).

EXAMPLE 1 Cloning of Human trkB and trkC Receptors

A. Generation of Human trkB and trkC Probes

Human brain cDNA, polyA+ RNA, genomic and cDNA libraries were obtainedfrom Clontech (Palo Alto).

In order to amplify fragments of the human trkB and trkC sequences foruse in probing cDNA libraries, the PCR with degenerate primers based onknown sequences of rat trkB or pig trkC (see Table 1), was employed. PCRreaction buffer consisted of 10 mM Tris pH 8.4 at room temperature, 2.0mM MgCl₂ and 50 mM KCl. A "hot start" procedure was used for allreactions, samples without enzyme were incubated for ten minutes at 98°C., equilibrated to 65° C. and enzyme added. They were then cycledthirty-five times through 94° C. for 45 seconds; 60° for 45 seconds; and72° C. for 60 seconds and a final extension at 72° C. for ten minutes.

Fragments amplified by this procedure were subcloned into pGEM vector(Promega, Madison, Wis.) and sequenced. Inserts from clones withsequences similar to known trkB and C. sequences were then excised,gel-purified and labeled by random priming with 32P dCTP. These wereused to probe 10⁶ cDNA clones which had been plated at 5×10⁴ plaques per15cm dish, transferred to nitrocellulose (Schleicher and Schuell, Keene,N.H.) in duplicate, denatured with alkali, neutralized and baked at 80°C. for two hours. Filters were prehybridized at 42° C. for at least fourhours in 50% formamide, 5× SSC, 5× Denhardt's, 20mM NaPO4, pH 7.0, 0.1%SDS, and 100 micrograms/ml salmon sperm DNA and hybridized overnight inthe same conditions with Denhardt's reduced to 1×. Filters were thenwashed four times in 2× SSC, 0.1%SDS and twice with 0.1× SSC, 0.1% SDSat room temperature and twice with 0.1× SSC, 0.1% SDS at 42° C. Cloneswhich were positive on both sets of filters were plaque purified and theinserts subcloned either by helper mediated excision (lambda DR2libraries) or by standard subcloning into PGEM. Oligonucleotide probeswere either end labeled using polynucleotide kinase or labeled by"fill-in" reactions using Klenow fragment of DNA polymerase andhybridized to filters under the same conditions but with formamidereduced to 35%. Genomic clones hybridizing to the 5' probe for trkB weredigested with Sau3a and resulting fragments were subcloned into BamHIcut M13 mp18. These clones were rescreened as for the lambda libraries(with no denaturation step) and positive clones were plaque purified andsequenced. DNA encoding the full coding region of trkB and trkC werereconstructed using standard techniques.

B. Characterization of Human trkB Clones

Six clones were obtained using the probe for human trkB. These weremapped using the PCR and primers designed from the sequence obtained inthe initial probe and the clones with the greatest 3' and 5' extent weresequenced. Sequence analysis revealed that these clones encoded aprotein highly homologous to rodent trkB which contained an entiretyrosine kinase domain and were intact to the 3' poly A+ tail, but wereapparently incomplete at the 5' end. An oligonucleotide probe designedfrom the 5' end of the rat trkB sequence was used to rescreen theinitial library and subsequently four other dT primed human brainlibraries with no positive clones found. Four positive clones wereobtained when a random primed human brain library was screened with thisprobe. Sequence analysis of these clones indicated that they overlappedwith the previous human clones, but, by comparison with the rat, werestill missing seventeen bases of coding region at the 5' end. A humangenomic library was then probed with the 5' oligonucleotide probe andgenomic clones isolated. Sau3a fragments of these clones were thensubcloned into M13, rescreened, and positive subclones were sequenced toobtain the last of the coding sequence. The final nucleotide and deducedamino acid sequence of human trkB obtained from the overlapping regionsof the cDNA clones is shown in FIGS. 1A-1C.

C. Characterization of Human trkC Clones

A similar strategy was used to generate probes specific for theextracellular domain of human trkC, and two initial clones wereobtained. Both of these were found to contain sequences corresponding tothe truncated form of trkC described in the pig and rat (Lamballe etal., 1991! supra; Tsoulfas, 1993! supra; Valenzuela et al., 1993!,supra), since the sequence encoded the complete extracellular domain oftrkC, a transmembrane domain and a short cytoplasmic domain whichcontained no TK-like sequences. In order to isolate clones encoding thetyrosine kinase domain of trkC, libraries were reprobed in duplicatewith oligonucleotides corresponding to the C-terminal tail of pig trkCand the juxtamembrane region of the intracellular domain of human trkC.Double positive clones were analyzed and found to contain sequenceoverlapping with the truncated trkC clones and also containing atyrosine kinase coding sequence. The nucleotide and deduced amino acidsequence obtained from the overlapping regions of these clones is shownin FIGS. 2A-2C.

D. Cloning of Human trkA

In addition, trk A was recloned from human brain with the PCR by usingexact match primers and human brain cDNA as a template. A resultingclone was sequenced, and five discrepancies with the previouslypublished sequence were seen. Each of these areas were examined bydirect sequencing of several different amplification reactions and trueerrors in the clone sequenced were corrected by site specificmutagenesis. There remained one difference with the previouslydetermined sequence, a GC for CG transposition leading to a switch fromserine to cysteine at residue 300 in the deduced amino acid sequence.Due to the sequencing of multiple reactions, and the conservation ofthis cysteine in rat trkA (Meakin et al., 1992!, supra) and all otherknown trks (see below), it seems likely that the original sequence is inerror.

E. Results

Examination of the sequences obtained from the human clones andcomparison to the known structure of rat and mouse trkB and rat and pigtrkC indicates that there is a very high degree of overall sequencesimilarity across these mammalian species. The overall structural motifsidentified by Schneider and Schweiger (1991), supra are maintained,namely, a signal sequence, predicted to be clipped at residues 31 forboth trkB and C (later confirmed by N-terminal sequence analysis, seeexpression of trk immunoadhesins), two cysteine rich domains flanking aleucine rich domain, two Ig like domains of the C2 type, a transmembranedomain, and a tyrosine kinase domain showing high similarity to otherknown tyrosine kinases. There are 11 and 13 potential N-linkedglycosylation sites in the extracellular domains of trkB and C,respectively. The similarity of different regions of the known trkswithin and across species is shown in FIG. 3.

During sequence analysis of several of the different clones obtained fortrkB and C, multiple forms apparently arising from alternate splicingwere seen. Variant forms were observed as a possible insert in theextracellular domain of trkC, truncated, non TK forms of trkB and C, anda possible insert within the TK domain of trkC. Using library screeningwith specific oligonucleotide probes, and the PCR, a more systematicsearch was then undertaken to search for potential other variants atthese sites in the different human trks. A diagram of the differentforms found in the different human trks and comparison to those found inother known trks is shown in FIG. 4.

In the extracellular domain of human trkC, there was a possible deletionof nine amino acids compared to rat and pig trkC at a site near to thatwhere the extracellular insert was described in rat and human trkA(Barker et al., J. Biol. Chem. 268, 1510-15157 1993!; FIG. 2). PCRanalysis of this region in human trkC showed only two bands,corresponding in length to that expected for the insert-containing andinsert-deleted forms. PCR analysis of this region in human trkB showedno detectable length polymorphisms, but amplification using trkAspecific primers did show two distinct bands which were cloned andsequenced. The potential nucleotide insert was TCTCCTTCTCGCCGGTGG (SEQ.ID. NO: 38) at position 1297 coding for the identical peptide insert(SEQ ID NO: 39) previously described in rat and human trkA (Barker, etal., supra).

From the human brain libraries, both trkB and C clones were obtainedwhich did not encode a TK domain but instead showed an alternate,truncated intracellular domain. In trkB, this consisted of eleven newamino acids added after position 435 which are identical to thosepreviously identified in the rat as t1 (Middlemas, et al., 1991, supra)and in the mouse as the truncated form (Klein et al., EMBO J. 8,3701-3709 1989!). All attempts using cDNA libraries probed witholigonucleotides or using PCR, failed to yield sequences from the humansimilar to those identified in the rat as t2 (Middlemas, et al. 1991!,supra). The PCR readily yielded sequences similar to t2 when eithermouse or rat brain cDNA was used as a template, showing that t2 is notunique to the rat and that the techniques used were capable of detectingt2 like sequences at least from the rodent (data not shown).

The truncated form of trkC was longer than that in trkB, and similar tothat previously described in pig trkC (Lamballe, et al. 1991!, supra)and in the rat (Tsoulfas et al., 1993!, supra) or as the ic158 form ofrat trkC (Valenzuela et al., 1993!, supra). This form consisted of 83additional amino acids starting at position 498, which were highlyconserved across species. In this span, there were only two differences,an aspartate to glutamate and a serine to proline substitution, seenacross all three species.

The TK domain of trkC obtained in the cDNA clones contained an apparentinsert of fourteen amino acids between subdomains VII and VIII (Hanks etal., Science 241: 42-52 1988! and Hanks et al., Methods in Enzymol. 200:38-62 1991!. This sequence is inserted in the same site as the observedpotential inserts seen in the rat trkC TK domain and is identical insequence to the fourteen amino acid insert Been there (Hanks et al.,1988!, supra; Valenzuela et al., 19933, supra). In addition to thefourteen amino acid insert seen in rat trkC, longer inserts oftwenty-five (Tsoulfas et al., 1993!, supra) or thirty-nine (Valenzuelaet al., 1993!, supra) amino acids have been seen. In an attempt todetermine if these longer inserts were expressed in the human, braincDNA was used as a template for PCR amplification across this region(see FIG. 5). These experiments consistently showed two bands of lengthscorresponding to the two already observed splice forms, i.e., with andwithout the fourteen amino acid insert. Cloning and sequencing of thesetwo bands verified that they correspond to the two forms with andwithout the previously seen fourteen residue insert. Interestingly, thissplicing was tissue specific as only the band corresponding to theinsert-free form was seen in amplifications using cDNA from a non-neuraltissue expressing high levels of trkC, the testis (data not shown). PCRof human brain cDNA using oligos specific for the same region of trkB TKdomain showed no evidence for length polymorphisms in this region (seeFIG. 5).

F. Discussion

By examining the degrees of similarity between the different trks in asingle species and the same trk in different species, certaingeneralizations may be drawn. The comparison of the three human trks toeach other and the equivalent trk from the rat is shown for thedifferent domains as defined by Schneider and Schweiger (1991), supra inFIG. 3. Each of the trks is quite conserved between human and rat, withtrkB and trkC being almost identical across these two mammalian species.Each individual domain of trk B and trkC is at least 85% similar betweenrat and human. On the other hand, trkA, although its overall degree ofsimilarity between human and rat is quite high, shows regions ofsignificant sequence divergence. In particular, in the extracellulardomain, it is only the leucine rich and second Ig-like domain which areat least 85% similar. This may have implications for the localization ofthe neurotrophin binding domain(s) of the trks. The transmembrane andintracellular domains of trkA are highly conserved between rat andhuman, similar to trkB and trkC. When similarity comparisons ofdifferent trks in the human are examined, it is readily apparent thatthe TK domain is the most highly conserved across the different trks. Ofthe extracellular domains, it is again the second Ig-like domain, alongwith the second cysteine rich domain which are most similar between thedifferent human trks.

In contrast to the conservation of sequence, were the observeddifferences between the human and previously known trks in the form ofdifferently processed transcripts. In the rodent, trkB contains at leasttwo different truncated forms and northern blots probed for trkB exhibita complex pattern with many transcript sizes. We failed to find evidencefor the existence of the t2 form in the human despite considerableeffort and observed a much simpler transcript pattern. for trkB. Whilewe cannot rule out the existence of a homolog of this form in the human,a t2 equivalent seems unlikely to be expressed as abundantly as in therodent.

One of the proposed roles for the truncated forms of the trks is to actas a dominant negative influence on signal transduction by neurotrophinin the expressing cell (Jing et al., Neuron 9, 1067-1079 1992!). This isconsistent with the relative lack of efficacy of neurotrophin signallingseen in tissue from the adult brain when stimulated by neurotrophins(Knusel et al., J. Neurosci. 1994!), as the ratio of truncated to nontruncated forms of the trks is quite high in the adult (see FIG. 6). Ifthis is the main role for truncated trks, then the apparent absence oft2 in the human is all the more interesting, as it has been shown that,in the rodent, t2 is primarily expressed in neurons, while the othertruncated form of trkB, t1, is primarily in non-neuronal cells. If thislocalization were also true in humans, then human neurons, without t2,would express a much lower level of truncated form of trkB relative torodents. Thus, the proposed dominant negative effect might not be asimportant in human neurons as in the rodent.

There are also differences between human and previously describedtranscripts of trk C. In the extracellular domain, there is apparentalternate splicing giving rise to two forms, with and without an insertof nine amino acids. This apparent insertion site aligns with thepreviously characterized insertion site in rat trkA. As yet, nofunctional differences in binding or signal transduction have beendetected between the two splice forms in the rat trkA where the insertis six amino acids (Barker et al., J Biol. Chem. 268, 1510-15157 1993!),but perhaps the there will be greater differences in the human trkCforms with a nine amino acid insert. Whatever the biological role forthe differently spliced forms, they are quite species specific, since noevidence of an insert in this location was seen in human trkB in thisstudy, and previous work has not detected the insert in trkC outside thehuman (Valenzuela et al. 1993!, supra; Tsoulfas, 1993!, supra; Lamballe,et al. 1991!, supra).

We also found examples of various forms of human trkC presumably due toalternate spicing in the intracellular part of the molecule. We observedthe presence of a truncated form of trkC, which does not contain any ofthe consensus tyrosine kinase domain. Unlike trkB, where the truncatedforms have a very short cytoplasmic tail, the cytoplasmic portion oftruncated human trkC is 83 residues long. In addition, there is a veryhigh degree of conservation among species in this region, suggestingthat it may have an important function, perhaps serving as a signal tospecify subcellular localization.

As has been described for rat trkC, there are forms of human trkC whichcontain an insert in the TK domain. Unlike the rat, where there arepossible inserts of fourteen and twenty-five or thirty-nine amino acids,there appears to be only a fourteen amino acid insert possible at thissite in the human. It is likely that these inserts play an importantrole in modulating the signalling cascade induced by ligand binding totrkC. Using PC12 cells expressing various forms of trkC as the assaysystem for signal transduction, it has been shown that expression oftrkC with no insert in the TK domain confers on the expressing cells theability to respond to NT3 with neurite outgrowth as well as NT3-inducedautophosphorylation. Cells expressing trkC containing a TK insert arecapable of ligand induced autophosphorylation, but do not respond to NT3with neurite outgrowth. There are no differences yet described betweenthe various inserts in this regard, but there are many downstreamsequelae to neurotrophin binding and very few have been examined todate. This processing is tissue specific, as no evidence of the fourteenresidue insert containing form was observed in human testis.

EXAMPLE 2 Expression Pattern of trk Receptors in Human Tissues

A. Northern Analysis

Probes used for Northern analysis were labeled using the PCR and theprimers indicated in Table 1 on appropriate cloned template DNA. PCRreactions were run as described for initial cloning except thatunlabeled dCTP was replaced in the reaction with gamma 32P dCTP at aconcentration of 8 mCi/ml (3,000 Ci/mmole) and the reaction was only runfor twenty cycles. Probes were separated from unincorporated nucleotidesand boiled for five minutes before being added to Nytran blotscontaining 2 micrograms of poly A+ RNA per lane (Clontech, Palo Alto,Calif.) which had been prehybridized in 5× SSPE, 10× Denhardt's,100ug/ml salmon sperm DNA, 50% formamide, and 2% SDS. Hybridization wascarried out at 50° C. in the same solution overnight and then blots werewashed as for library filters but with the final wash at 50° C.Autoradiograms were obtained using a Fuji BAS2000 image analyzer afterexposing the imaging plate for ten to twenty hours.

Results

The expression pattern and transcript size of the trks in human tissueswas examined by using Northern analysis (FIG. 6).

Hybridization with probes for trkB yielded an apparently simple pattern,with a transcript of 6.9 kb hybridizing to both an extracellular and TKspecific probes, and a transcript of 8.1 kb hybridizing only to the TKspecific probe. On the basis of this simple result, the 8.1 kbtranscript presumably corresponds to the full length, TK-containingmessage, while the 6.9 kb transcript corresponds to message encoding thesingle truncated form seen in human. As might be expected from thegreater number of potential splice variants detected while cloning trkC,probing Northerns for this molecule led to a more complex pattern ofhybridization. Transcripts of 11.7, 7.9 and 4.9 kb were detected with aprobe specific for the TK domain, while an additional transcript of 4.4kb was detected with the extracellular domain probe (see FIG. 6).

Of the human tissues examined, both trkB and trkC were expressed ingreatest abundance in the brain. However, there was expression in avariety of locations outside the nervous system in both adult and fetaltissues. The 8.1 kb transcript of trkB containing the TK domain wasexpressed in kidney, skeletal muscle and pancreas, while in heart,spleen and ovary expression of only the truncated form was detected. Infetal tissues, TK containing trk B was found not only in brain, but alsoin kidney and lung, while truncated trkB was found in brain, kidney,lung and heart. It was apparent that the ratio of TK-containing totruncated trkB transcripts was much higher in fetal than adult brain.

Although the highest expression level of trkC was in brain, there waswidespread expression of trkC outside the nervous system. In the adult,TK containing trkC was expressed in kidney, skeletal muscle, lung,heart, small intestine, ovary, testis, and prostate, while in the fetus,the greatest expression was in brain, kidney, lung, and heart. The 4.4kb transcript corresponding to the truncated form of trkC was detectedin all tissues examined except peripheral blood leukocytes. Similar tothe case for trkB, the ratio of TK containing to truncated trkC washigher in fetal compared to adult brain.

Discussion

Analysis of the transcripts for trkB using Northern blots showed arelatively simple pattern compared to that seen in the rodent. This isconsistent with the idea that there is only a single main truncated formof trkB in the human. Analysis of the trk C showed a more completepattern of transcript sizes, in keeping with the greater number of formsdetected during sequence analysis of the clones. No evidence was seenfor a transcript hybridizing with the kinase probe but not with theextracellular probe as has been described in rat trkC Valenzuela et al.,1993!, supra). In analyzing different tissues, the primary location oftrkB and trkC expression was in the nervous system and specifically inthe regions of the CNS. Unexpected was the finding that there is lowlevel expression of trkB and trkC in a wide variety of tissues outsidethe nervous system. The levels of expression were quite low compared tothose found in various regions of the brain, but still quite detectableabove background. Some of the expression seen in certain tissues may bedue to expression on elements of the nervous system sparsely scatteredthrough the tissue. For example, expression of trkC in the smallintestine may turn out to be due in whole or in part to expression bythe neurons of the enteric nervous system. Final elucidation of thiswill have to await a detailed in situ hybridization analysis of tissuesoutside the nervous system.

B. In Situ Hybridization

In situ hybridization was carried out by a modification of a previouslypublished procedure (Phillips et al., Science 250, 290-294 1990!).Tissue was prepared for hybridization by a variety of techniques.Autolysis times on all samples were under 24 hours. Whole, unfixedembryos were embedded in OCT, frozen by floating the blocks in petridishes on liquid nitrogen, and sectioned with the aid of a cryostat.Sections were thaw-mounted onto slides (superfrost plus, Fisher),air-dried, baked at 55° C. for 10", and stored in sealed boxes withdesiccant at -70° C. until use. Adult dorsal root ganglia were fixed byimmersion in 4% formaldehyde and either processed for paraffinsectioning or for crysosectioning. Brain specimens were fixed byimmersion for 24 hours in 4% formaldehyde, cryoprotected for 24 hours inbuffered sucrose, frozen on dry ice, and cut on a freezing slidingmicrotome. Sections were stored (less than 48 hours) in phosphatebuffered saline at 4° C., mounted onto gelatin-subbed slides, air-dried,and stored at 4° C. Care was taken to avoid any condensation of moistureon all tissue sections during storage of the tissue.

On the day of hybridization, tissue sections were differentiallypretreated according to the fixation and sectioning protocol employed togenerate the sections. Unfixed tissue sections were fixed by immersionin 4% formaldehyde, 1% glutaraldehyde in 0.1M sodium phosphate for 30"at 4° C., rinsed in 0.5× SSC (20× SSC is 3M NaCl and 0.3M sodiumcitrate), and placed directly into prehybridization solution.Cryosections of immersion-fixed tissue were fixed in 4% formaldehyde in0.1M sodium phosphate for 5 minutes, rinsed O.5× SSC, digested for 30minutes at room temperature with proteinase-K (Boehringer-Manheim; 25μg/ml in 0.5M NaCl and 10 mM Tris, pH 8.0), rinsed, refixed for 10minutes in 4% formaldehyde, dehydrated in a series of alcohols (50%ethanol containing 0.3t ammonium acetate; 70% ethanol containingammonium acetate; 100% ethanol; 2 minutes per incubation), rehydratedthrough the same series of ethanols, and rinsed again in 0.5× SSC priorto prehybridization. For paraffin-embedded tissue, deparaffinzation wasperformed by 2 rinses in xylene (2 " each), after which tissue wasrehydrated through a series of alcohol solutions (100% ethanol twice,95% ethanol, 70% ethanol; 2" each). Tissue sections were then fixed in4% formaldehyde for 10", digested for 30" with proteinase k (25 or 50ug/ml ; room temperature or 37° C.), rinsed, refixed for 10", and rinsedagain in 0.5× SSC prior to prehybridization.

Prehybridization, hybridization, and posthybridization RNAase treatmentand stringency washes were identical for all tissues carried out aspreviously described (Phillips et al, 1990).

In situ hybridization with probes to human trkA, and the TK-containingforms of trkB, and trkC was conducted on a limited series of embryonicand adult human tissue prepared by a variety of protocols. In twoembryos of 6 & 8 weeks gestation (fresh-frozen), trkA expression wasrestricted to dorsal root and cranial sensory ganglia, including thetrigeminal ganglion (FIG. 7A). In contrast, trkB and trkC were not onlyexpressed in sensory ganglia, but prominent expression was also seenwithin the developing brain and spinal cord (FIG. 7B & C). In addition,trkC expression was observed in the developing vasculature.

Results

Within developing dorsal root ganglia, trkC was strongly expressed inganglia from both the 6 and 8 week embryos. Curiously, in both embryos,there was a marked tendency for trkC-expressing cells to localize in theventral end of the ganglia (FIGS. 8A-8F). In contrast, trkA positivecells were largely restricted to dorsal portions of the ganglia (FIGS.8A-8F). In adult dorsal root ganglia (paraffin-embedded or cryosectionedfixed tissue), a subpopulation of DRG neurons was labelled with each ofthe three trk probes (trkB, FIGS. 9B & C; trkA and C data not shown).Cells labelled with probes to each of the three trks appeared to berandomly distributed throughout the ganglia. No labelling ofnon-neuronal cells was observed with any of the probes.

In the adult human forebrain (fixed, cryosectioned tissue), cellsstrongly labelled for trkA expression were observed in the nucleusbasalis of Meynert and scattered throughout the head of the caudatenucleus (FIG. 7D). Labelled cells were of large diameter and conform tothe expected appearance of cholinergic cells (FIG. 9A). trkC was widelyexpressed throughout the human forebrain, including prominent expressionin hippocampus and neocortex (FIGS. 7E; 9D & E) and labelled cellsappeared to be exclusively of neuronal morphology (FIGS. 9A-9G).

Discussion

The in situ hybridization analysis of the expression of the members ofthe trk family in the human nervous system confirmed that the overallexpression pattern is similar to that seen in other mammals. This shouldprovide a foundation for further studies designed to examine theexpression of the differently spliced forms of the human trks in detailin certain areas of normal and pathological tissues. In this regard,given the difficulty of obtaining human tissue, it is encouraging thatthe in situ hybridization was performed on tissues handled in a varietyof ways post mortem. Sections were cut unfixed, fixed and frozen, andfixed and paraffin-embedded, and all of these methods yielded usefulresults. One unexpected finding was the apparent polarization of thedeveloping human DRG, with trkA cells predominant in the dorsal and trkCexpressing cells predominant in the ventral area of the developingganglia. This polarization of trk expression was not apparent insections from the adult human DRG or in rat embryos hybridized with rattrkA and trkC probes (data not shown).

EXAMPLE 3 Expression of trk Immunoadhesins

A. Construction of trk-Ig Immunoadhesins

Using protein engineering techniques, the human trks were expressed aschimeras of trk extracellular domain with the Fc domain of human IgGheavy chain. DNA constructs encoding the chimeras of trk extracellulardomain and IgG-1 Fc domains were made with the Fc region clones of humanIgG-1 (Ashkenazi et al., Immunoadhesins Intern, Rev. Immunol. 10,219-227 1993!). More specifically, the source of the IgG-1 encodingsequence was the CD4-IgG-1 expression plasmid pRKCD4₂ Fc₁ (Capon et al.,Nature 334, 525 1989!; Byrn et al., Nature 334, 667 1990!) containing acDNA sequence encoding a hybrid polypeptide consisting of residues 1-180of the mature human CD4 protein fused to human IgG-1 sequences beginningat aspartic acid 216 (taking amino acid 114 as the first residue of theheavy chain constant region (Kabat et al., Sequences of Proteins ofImmunological Interest 4th ed. 1987!), which is the first residue of theIgG-1 hinge after the cysteine residue involved in heavy-light chainbonding, and ending with residues 441 to include the CH2 and CH3 Fcdomains of IgG-1.

The CD4-encoding sequence was deleted from the expression plasmidpRKCD4₂ Fc₁ and the vector was fused to DNA encoding the trk receptors,with the splice between aspartate 216 of the IgG-1 and valine 402 oftrkA, threonine 423 of trkB numbering from the translation startmethionine, or threonine 413 of trkC insert-deleted variant, numberingfrom the translation start methionine. DNAs encoding whole receptor orIgG chimeras were subcloned into pRK for transient expression in 293cells using calcium phosphate (Suva et al., Science 237, 893-896 1987!).For purification of trk-IgG chimeras, cells were changed to serum freemedia the day after transfection and media collected after a further twoto three days. Media was filtered, bound to a protein A column (Hi-TrapA, Pharmacia), the column washed with PBS, bound protein eluted with0.1M glycine, pH 3.0, and immediately neutralized with tris buffer.Concentration was estimated by absorbance at 280 nm using an extinctioncoefficient of 1.5. SDS-PAGE analysis showed the resulting protein to bea single detectable band.

Cells transiently transfected with these DNA constructs secreted proteinwhich bound to protein A and migrated with an approximate molecularweight of 125 kD on reducing SDS-polyacrylamide gels. Purified trk-IgGchimeras could be easily isolated from conditioned media in a singleround of affinity chromatography on a protein A column. Sequenceanalysis of these purified proteins verified the predicted signalsequence cleavage site, and resulting N-termini (data not shown).

B. Binding Assays

In order to test whether these chimeric proteins retained the bindingspecificity expected of the trk extracellular domain in a cellularenvironment, competitive displacement assays were done with iodinatedneurotrophins. As can be seen from the results shown in FIG. 10, thetrk-IgG chimeras did show specific binding to the expectedneurotrophin(s). Chimeras containing trkA extracellular domain bound NGFwell and NT3 and NT5 with much lower affinity. Chimeras containing trkBbound BDNF and NTS well but only slightly better than NT3, and showedalmost no detectable binding to NGF. Chimeras containing trkC werehighly specific for NT3 over the other neurotrophins. The apparentaffinity of the chimeras for their preferred ligand as determined inthese competitive displacement assays is in the range of that determinedfor the majority of the binding sites on cells transfected with andexpressing the various trk proteins. In one experiment, the IC50sobtained for trkA were 62 pM for NGF and 20 nM for NT3, for trkB were 81pM for BDNF, 200 pM for NT4/5 and 18 nM for NT3 and for trkC was 95 pMfor NT3. The ratio of specific to nonspecific binding are quite high inassays done with these reagents, usually at least ten to one (see FIGS.10A-10C).

To check whether the trk-IgG chimeras might be capable of blocking thebiological activity of their cognate ligands, the neurotrophin inducedsurvival of peripheral neurons was assayed in the presence of theappropriate trk-IgG chimera. As can be seen in FIGS. 11A-11C, trkA-IgGis a potent inhibitor of NGF biological activity, trkB-IgG of BDNF, andtrkC-IgG of NT3. In all cases, addition of excess neurotrophin iscapable of overcoming this blockade, indicating that the trk-IgGchimeras are not generally toxic to the neurons.

The binding data presented here demonstrates that the trk-IgG fusionsbind neurotrophins with a selectivity and affinity similar to that seenby expression of the whole receptor in cells. The binding assays asreported here are very simple to do in large numbers, have excellentreproducibility and low background, and retain the specificity of thenative trks. These qualities have proven quite valuable in analyzing thebinding characteristics of mutant neurotrophins (Laramee et al., Highresolution mapping-of NGF-trkA and p75 receptor interactions bymutagenesis.

In addition to their utility in analyzing the binding of neurotrophins,the trk-IgG chimeras are useful inhibitors of the biological activity oftheir cognate neurotrophin. All of the experiments shown here have beenperformed in in vitro systems, but preliminary experiments indicate thattrkA-IgG is capable of inhibiting NGF activity in vivo as well (data notshown). This will fill an unmet need for the trkB and trkC chimeras, asit has been difficult to raise good blocking antisera to BDNF, NT3 andNT4/5.

With the information in hand about the forms of trk present in human, itis possible to begin to investigate the expression of these forms in thenormal and diseased state. Knowledge of the expression levels of theentire spectrum of forms of each trk will be crucial, as the differentforms can display different and sometimes counteracting signaltransduction properties in response to neurotrophins. In addition, theavailability of soluble forms of the human trks should, by allowing theblocking of endogenous bioactivity, accelerate the investigation of thebiology of neurotrophins in vivo.

EXAMPLE 4 Mutagenesis of Human trkC

Mutagenesis studies were performed in order to determine which aminoacids of the extracellular domain of the trkC protein determine affinityand specificity to the neurotrophin NT-3. The three-dimensionalstructure of trkC is unknown, however, a putative domain organizationwas proposed. According to this model, the extracellular domains of thetrk family of proteins are built up by five domains. Proceeded by asignal sequence, the domains are: a first cysteine-rich domain, aleucine-rich domain, a second cystein-rich domain, and twoimmunoglobulin-like domains.

In order to investigate the function of the trkC receptor domains, fivetrkC variants were constructed, lacking each of the five domainsindividually (Δ1-Δ5) and one variant where all domains except the secondimmunoglobulin-like domain are deleted (Δ6). The structures areillustrated in FIG. 12. In addition to these variants, also all fivedomains were exchanged individually by the corresponding trkB sequence(s1-s5) in order to determine the remaining affinity to NT-3 and to testfor recruitment of BDNF binding. All trkC variants, including the trkC,trkB chimeras, were studied in the form of immunoadhesins. Theimmunoadhesins were constructed on the analogy of the process describedin Example 3, and expressed in the human embryonic kidney cell line 293,using a pRK5 (EP 307,247) or pRK7 vector. pRK7 is identical to pRK5except that the order of the endonuclease restriction sites in thepolylinker region between ClaI and HindIII is reversed. (See U.S. Pat.No. 5,108,901 issued Apr. 28, 1992). The proteins were secreted intoserum free medium, 20× concentrated and quantified with an anti-Fc ELISAassay. The results of a typical expression are presented in FIG. 13.Variants of particular interest, trkC, Δ6, Δ5, s5 and trkB were purifiedto homogeneity over Protein A using standard protocols. The N-terminalsequences of these variants were determined and were as predicted.

All receptor variants were tested for their ability to bind labeled NT-3in competitive displacement assays using standard immunoadhesiontechnology. All the fusions and swaps were still able to bind NT-3 withsimilar affinity as trkC with the exception of Δ5. Although the totalbound labeled NT-3 for several variants was low (i.e. Δ1, Δ4, Δ2), theIC-50 values were all close to the trkC value (FIGS. 14A and 14B). Mostimportantly, the variant Δ6, which lacks all but the secondimmunoglobulin-like domain, retained most of the binding capability ofthe trkC full length receptor. In addition, deletion of this domain inΔ5 leads to a molecule that is not able to bind NT-3 at all (FIG. 14C).

All receptor variants were tested for their ability to bind labeled BDNFin competitive diplacement assays using the same type of assay as forthe NT-3 binding. Note that trkC is not able to bind BDNF. All variantsbut one failed to bind BDNF (FIGS. 15A-C). The only variant which boundBDNF was swap5 where the second immunoglobulin-like domain of trkC isexchanged by the one of trkB (FIG. 15C). This variant bound BDNF as wellas the trkB full length receptor.

The paramount importance of the second immunoglobulin-like domain forthe function of trkC and trkB is apparent from the foregoing results.Deletion of all but this domain retained essentially the full bindingcapacity of trkC. Deletion of this domain removed the ability of trkC tobind NT-3. Exchanging this domain created a trkC variant that was ableto bind BDNF with similar affinity as trkB.

EXAMPLE 5 Use of trkA-IgG Immunoadhesin in the Treatment of InflammatoryPain

A. Blocking of Carageean-induced Hyperalgesia in Rats

50 μl of a 2% aqueous solution of carageenan (Sigma, Lot # 21H0322)alone or in combination with 15 μg of the trkA-IgG chimera prepared inExample 3 was injected into one hind paw of four adult male Wistar ratsat time zero. The latency of withdrawal to a noxious heat stimulus wasmeasured for each hind paw in triplicate every two hours thereafter. Thepaw injected with carageenan alone showed distinct inflammation andhyperalgesia (decreased latency to withdrawal compared to contralateralcontrol paw) within two hours. Rats injected with carageenan plustrkA-IgG showed distinct inflammation, but showed no evidence ofhyperalgesia compared to the contralateral control paw. Pooled data fromcarageenan alone vs. carageenan plus trkA-IgG at four, six and eighthour time points is significantly different at p>0.02 (see FIG. 17).

B. trkA-IgG Immunoadhesin Leads to Hypoalgesia

The trkA-IgG immunoadhesin was infused continuously under the skin ofthe dorsolateral surface of one hind paw of four adult male Wistar ratsat a rate of 0.5 μg/hr. Latency of withdrawal of control and infusedpaws was determined in triplicate at various times thereafter. Afterfive days of infusion, there was a pronounced hypoalgesia on the infusedside when compared to the control side. Withdrawal time difference ofall time points five days and after significantly differed from thepooled preinfusion time difference at p>0.05 (see FIG. 18).

                                      TABLE 1    __________________________________________________________________________    USE   trkA           trkB              trkC    __________________________________________________________________________    degenerate           TGYGAYATHATGTGGYTNAARAC                                           TGGATGCARYTNTGGCARCARCA    sense                SEQ. ID. NO: 10   SEQ. ID. NO: 11    degenerate           YTCRTCYTTNCCRTAYTCRTT                                           CCYTCYTGRTARTAYTCNACGTG    anti                 SEQ. ID. NO: 12   SEQ. ID. NO: 13    ECD insert          CACGTCMCMCGGCMCTACA                         GGAAGGATGAGAMCAGATTTCTGC                                           CATCMTGGCCACTTCCTCMGG    sense SEQ. ID. NO: 14                         SEQ. ID. NO: 15   SEQ. ID. NO: 16    ECD insert          AGGTGTTTCGTCCTTCTTCTCC                         GAGATGTGCCCGACCGGTTGTATC                                           CACAGTGATAGGAGGTGTGGGA    anti  SEQ. ID. NO: 17                         SEQ. ID. NO: 18   SEQ. ID. NO: 19    TK insert          GGATGTGGCTCCAGGCCCC                         GGGCMCCCGCCCACG#AA                                           ACGCCAGGCCAAGGGTGAG    sense SEQ. ID. NO: 20                         SEQ. ID. NO: 21   SEQ.ID. NO: 22    TK insert          TAACCACTCCCAGCCCCTGG                         TTGGTGOCCTCCAGCGGCAG                                           MTTCATGACCACCAGCCACCA    anti  SEQ. ID. NO: 23                         SEQ. ID. NO: 24   SEQ. ID. NO. 25    Probes    ECD sense          GCTCCTCGGGACTGCGATGC                         ATGTCGCCCTGGCCGAGGTGGCAT                                           AAGCTCAACAGCCAGMCCTC          (SEQ. ID. NO: 26)                         (SEQ. ID. NO: 27) (SEQ. ID. NO. 28)    ECD anti          CAGCTCTGTGAGGATCCAGCC                         CCGACCGGTTTTATCAGTGAC                                           ATGATCTTGGACTCCCGCAGAGG          (SEQ. ID. NO: 29)                         (SEQ. ID. NO: 30) (SEQ. ID. NO. 31)    TK specific          CTTGGCCAAGGCATCTCCGGT                                           ATGTGCAGCACATTMGAGGA    sense                (SEQ. ID. NO: 32) (SEQ. ID. NO. 33)    TK specific          TTATACACAGGCTTAAGCCATCCA                                           AGGAGGCATCCAGCGMTG    anti                 (SEQ. ID. NO: 34) (SEQ. ID. NO. 35)    __________________________________________________________________________

The entire disclosures of all citations cited throughout thespecification, and the references cited therein, are hereby expresslyincorporated by reference.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those ordinarily skilled in the art that various modificationsmay be made to the disclosed embodiments without diverting from theoverall concept of the invention. All such modifications are intended tobe within the scope of the present invention.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 41    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 3194 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (ii) MOLECULE TYPE: nucleic acid    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    #              50AAGAAGC CGCAAAGCGC AGGGAAGGCC TCCCGGCACG    #              100GCCGGT GCAGCGCGGG GACAGGCACT CGGGCTGCA    #              150TGTCGT CCTGGATAAG GTGGCATGGA CCCGCCATG    #              200TTCTGC TGGCTGGTTG TGGGCTTCTG GAGGGCCGC    #             250CGTCCTG CAAATGCAGT GCCTCTCGGA TCTGGTGCAG    #             300GGCATCG TGGCATTTCC GAGATTGGAG CCTAACAGTG    #             350CATCACC GAAATTTTCA TCGCAAACCA GAAAAGGTTA    #             400AAGATGA TGTTGAAGCT TATGTGGGAC TGAGAAATCT    #             450TCTGGAT TAAAATTTGT GGCTCATAAA GCATTTCTGA    #             500GCAGCAC ATCAATTTTA CCCGAAACAA ACTGACGAGT    #             550ATTTCCG TCACCTTGAC TTGTCTGAAC TGATCCTGGT    #             600ACATGCT CCTGTGACAT TATGTGGATC AAGACTCTCC    #             650CAGTCCA GACACTCAGG ATTTGTACTG CCTGAATGAA    #             700TTCCCCT GGCAAACCTG CAGATACCCA ATTGTGGTTT    #             750CTGGCCG CACCTAACCT CACTGTGGAG GAAGGAAAGT    #             800CTGTAGT GTGGCAGGTG ATCCGGTTCC TAATATGTAT    #             850ACCTGGT TTCCAAACAT ATGAATGAAA CAAGCCACAC    #             900AGGATAA CTAACATTTC ATCCGATGAC AGTGGGAAGC    #             950GGCGGAA AATCTTGTAG GAGAAGATCA AGATTCTGTC    #            1000ATTTTGC ACCAACTATC ACATTTCTCG AATCTCCAAC    #            1050TGGTGCA TTCCATTCAC TGTGAAAGGC AACCCAAAAC    #            1100GTTCTAT AACGGGGCAA TATTGAATGA GTCCAAATAC    #            1150TACATGT TACCAATCAC ACGGAGTACC ACGGCTGCCT    #            1200CCCACTC ACATGAACAA TGGGGACTAC ACTCTAATAG    #            1250TGGGAAG GATGAGAAAC AGATTTCTGC TCACTTCATG    #            1300TTGACGA TGGTGCAAAC CCAAATTATC CTGATGTAAT    #            1350GGAACTG CAGCGAATGA CATCGGGGAC ACCACGAACA    #            1400CCCTTCC ACAGACGTCA CTGATAAAAC CGGTCGGGAA    #            1450ATGCTGT GGTGGTGATT GCGTCTGTGG TGGGATTTTG    #            1500CTGTTTC TGCTTAAGTT GGCAAGACAC TCCAAGTTTG    #            1550AGCCTCC GTTATCAGCA ATGATGATGA CTCTGCCAGC    #            1600TCTCCAA TGGGAGTAAC ACTCCATCTT CTTCGGAAGG    #            1650GTCATTA TTGGAATGAC CAAGATCCCT GTCATTGAAA    #            1700TGGCATC ACCAACAGTC AGCTCAAGCC AGACACATTT    #            1750AGCGACA TAACATTGTT CTGAAAAGGG AGCTAGGCGA    #            1800AAAGTGT TCCTAGCTGA ATGCTATAAC CTCTGTCCTG    #            1850CTTGGTG GCAGTGAAGA CCCTGAAGGA TGCCAGTGAC    #            1900ACTTCCA CCGTGAGGCC GAGCTCCTGA CCAACCTCCA    #            1950GTCAAGT TCTATGGCGT CTGCGTGGAG GGCGACCCCC    #            2000TGAGTAC ATGAAGCATG GGGACCTCAA CAAGTTCCTC    #            2050CTGATGC CGTGCTGATG GCTGAGGGCA ACCCGCCCAC    #            2100TCGCAGA TGCTGCATAT AGCCCAGCAG ATCGCCGCGG    #            2150GGCGTCC CAGCACTTCG TGCACCGCGA TTTGGCCACC    #            2200TCGGGGA GAACTTGCTG GTGAAAATCG GGGACTTTGG    #            2250GTGTACA GCACTGACTA CTACAGGGTC GGTGGCCACA    #            2300TCGCTGG ATGCCTCCAG AGAGCATCAT GTACAGGAAA    #            2350GCGACGT CTGGAGCCTG GGGGTCGTGT TGTGGGAGAT    #            2400AAACAGC CCTGGTACCA GCTGTCAAAC AATGAGGTGA    #            2450TCAGGGC CGAGTCCTGC AGCGACCCCG CACGTGCCCC    #            2500AGCTGAT GCTGGGGTGC TGGCAGCGAG AGCCCCACAT    #            2550AAGGGCA TCCATACCCT CCTTCAGAAC TTGGCCAAGG    #            2600CCTGGAC ATTCTAGGCT AGGGCCCTTT TCCCCAGACC    #            2650GTACTCC TCAGACGGGC TGAGAGGATG AACATCTTTT    #            2700GGCCACC AAGCTGCTCT CCTTCACTCT GACAGTATTA    #            2750CGAGAAG CTCTCGAGGG AAGCAGTGTG TACTTCTTCA    #            2800TATTGAC TTCTTTTTGG CATTATCTCT TTCTCTCTTT    #            2850TTGTTCC TTTTTCTTTT TTTAAATTTT CTTTTTCTTC    #            2900TCCCTGC TTCACGATTC TTACCCTTTC TTTTGAATCA    #            2950ATTACTA TTAACTCTGC ATAGACAAAG GCCTTAACAA    #            3000TATCAGC AGACACTCCA GTTTGCCCAC CACAACTAAC    #            3050ATTCCTG CCTTTGATGT GGATGAAAAA AAGGGAAAAC    #            3100AAACTTT GTCACTTCTG CTGTACAGAT ATCGAGAGTT    #            3150TTCTATT TATTTATTAT TATTACTGTT CTTATTGTTT    #                 319 - #4ATAAAAAAA AAAAAAAATC TAGA    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 822 amino              (B) TYPE: Amino Acid              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    #Ala Met Ala Arg Leule Arg Trp His Gly Pro    #15    #Trp Arg Ala Ala Pherp Leu Val Val Gly Phe    #30    #Ser Arg Ile Trp Cyser Cys Lys Cys Ser Ala    #45    #Pro Arg Leu Glu Proro Gly Ile Val Ala Phe    #60    #Ile Phe Ile Ala Asnro Glu Asn Ile Thr Glu    #75    #Asp Val Glu Ala Tyrlu Ile Ile Asn Glu Asp    #90    #Ser Gly Leu Lys Phesn Leu Thr Ile Val Asp    #95                 1 - #00                 1 - #05    #Asn Leu Gln His Ilela Phe Leu Lys Asn Ser    #                120    #Ser Arg Lys His Phesn Lys Leu Thr Ser Leu    #                135    #Val Gly Asn Pro Pheeu Ser Glu Leu Ile Leu    #                150    #Thr Leu Gln Glu Alasp Ile Met Trp Ile Lys    #                165    #Cys Leu Asn Glu Sersp Thr Gln Asp Leu Tyr    #                180    #Ile Pro Asn Cys Glyro Leu Ala Asn Leu Gln    #                195    #Leu Thr Val Glu Glusn Leu Ala Ala Pro Asn    #                210    #Ala Gly Asp Pro Valhr Leu Ser Cys Ser Val    #                225    #Val Ser Lys His Metrp Asp Val Gly Asn Leu    #                240    #Arg Ile Thr Asn Ileis Thr Gln Gly Ser Leu    #                255    #Cys Val Ala Glu Asner Gly Lys Gln Ile Ser    #                270    #Leu Thr Val His Phesp Gln Asp Ser Val Asn    #                285    #Thr Ser Asp His Hishr Phe Leu Glu Ser Pro    #                300    #Pro Lys Pro Ala Leuhe Thr Val Lys Gly Asn    #                315    #Glu Ser Lys Tyr Ilesn Gly Ala Ile Leu Asn    #                330    #Glu Tyr His Gly Cysis Val Thr Asn His Thr    #                345    #Asn Gly Asp Tyr Thrsn Pro Thr His Met Asn    #                360    #Glu Lys Gln Ile Sersn Glu Tyr Gly Lys Asp    #                375    #Asp Gly Ala Asn Proly Trp Pro Gly Ile Asp    #                390    #Gly Thr Ala Ala Asnal Ile Tyr Glu Asp Tyr    #                405    #Glu Ile Pro Ser Thrhr Thr Asn Arg Ser Asn    #                420    #Leu Ser Val Tyr Alays Thr Gly Arg Glu His    #                435    #Cys Leu Leu Val Metla Ser Val Val Gly Phe    #                450    #Lys Phe Gly Met Lysys Leu Ala Arg His Ser    #                465    #Asp Ser Ala Ser Proal Ile Ser Asn Asp Asp    #                480    #Pro Ser Ser Ser Gluer Asn Gly Ser Asn Thr    #                495    #Thr Lys Ile Pro Valla Val Ile Ile Gly Met    #                510    #Asn Ser Gln Leu Lysln Tyr Phe Gly Ile Thr    #                525    #His Asn Ile Val Leual Gln His Ile Lys Arg    #                540    #Lys Val Phe Leu Alaly Glu Gly Ala Phe Gly    #                555    #Lys Ile Leu Val Alaeu Cys Pro Glu Gln Asp    #                570    #Ala Arg Lys Asp Pheys Asp Ala Ser Asp Asn    #                585    #Gln His Glu His Ilelu Leu Leu Thr Asn Leu    #                600    #Asp Pro Leu Ile Metly Val Cys Val Glu Gly    #                615    #Asn Lys Phe Leu Arget Lys His Gly Asp Leu    #                630    #Glu Gly Asn Pro Prosp Ala Val Leu Met Ala    #                645    #Ile Ala Gln Gln Ileln Ser Gln Met Leu His    #                660    #His Phe Val His Argal Tyr Leu Ala Ser Gln    #                675    #Glu Asn Leu Leu Valrg Asn Cys Leu Val Gly    #                690    #Val Tyr Ser Thr Asphe Gly Met Ser Arg Asp    #                705    #Pro Ile Arg Trp Metly Gly His Thr Met Leu    #                720    #Thr Thr Glu Ser Asple Met Tyr Arg Lys Phe    #                735    #Ile Phe Thr Tyr Glyly Val Val Leu Trp Glu    #                750    #Glu Val Ile Glu Cysyr Gln Leu Ser Asn Asn    #                765    #Arg Thr Cys Pro Glnrg Val Leu Gln Arg Pro    #                780    #Gln Arg Glu Pro Hiseu Met Leu Gly Cys Trp    #                795    #Leu Leu Gln Asn Leule Lys Gly Ile His Thr    #                810    #Leu Glyys Ala Ser Pro Val Tyr Leu Asp Ile    #                820    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1870 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #              50AAGAAGC CGCAAAGCGC AGGGAAGGCC TCCCGGCACG    #             100GGCCGGT GCAGCGCGGG GACAGGCACT CGGGCTGGCA    #             150ATGTCGT CCTGGATAAG GTGGCATGGA CCCGCCATGG    #             200CTTCTGC TGGCTGGTTG TGGGCTTCTG GAGGGCCGCT    #             250CGTCCTG CAAATGCAGT GCCTCTCGGA TCTGGTGCAG    #             300GGCATCG TGGCATTTCC GAGATTGGAG CCTAACAGTG    #             350CATCACC GAAATTTTCA TCGCAAACCA GAAAAGGTTA    #             400AAGATGA TGTTGAAGCT TATGTGGGAC TGAGAAATCT    #             450TCTGGAT TAAAATTTGT GGCTCATAAA GCATTTCTGA    #             500GCAGCAC ATCAATTTTA CCCGAAACAA ACTGACGAGT    #             550ATTTCCG TCACCTTGAC TTGTCTGAAC TGATCCTGGT    #             600ACATGCT CCTGTGACAT TATGTGGATC AAGACTCTCC    #             650CAGTCCA GACACTCAGG ATTTGTACTG CCTGAATGAA    #             700TTCCCCT GGCAAACCTG CAGATACCCA ATTGTGGTTT    #             750CTGGCCG CACCTAACCT CACTGTGGAG GAAGGAAAGT    #             800CTGTAGT GTGGCAGGTG ATCCGGTTCC TAATATGTAT    #             850ACCTGGT TTCCAAACAT ATGAATGAAA CAAGCCACAC    #             900AGGATAA CTAACATTTC ATCCGATGAC AGTGGGAAGC    #             950GGCGGAA AATCTTGTAG GAGAAGATCA AGATTCTGTC    #            1000ATTTTGC ACCAACTATC ACATTTCTCG AATCTCCAAC    #            1050TGGTGCA TTCCATTCAC TGTGAAAGGC AACCCAAAAC    #            1100GTTCTAT AACGGGGCAA TATTGAATGA GTCCAAATAC    #            1150TACATGT TACCAATCAC ACGGAGTACC ACGGCTGCCT    #            1200CCCACTC ACATGAACAA TGGGGACTAC ACTCTAATAG    #            1250TGGGAAG GATGAGAAAC AGATTTCTGC TCACTTCATG    #            1300TTGACGA TGGTGCAAAC CCAAATTATC CTGATGTAAT    #            1350GGAACTG CAGCGAATGA CATCGGGGAC ACCACGAACA    #            1400CCCTTCC ACAGACGTCA CTGATAAAAC CGGTCGGGAA    #            1450ATGCTGT GGTGGTGATT GCGTCTGTGG TGGGATTTTG    #            1500CTGTTTC TGCTTAAGTT GGCAAGACAC TCCAAGTTTG    #            1550TGTTTTG TTTCATAAGA TCCCACTGGA TGGGTAGCTG    #            1600GACAGAG AAAGGGGCTG TGGTGCTTGT TGGTTGATGC    #            1650GGACTCC TGGGACTGCT GTTGGCTTAT CCCGGGAAGT    #            1700GGTTTTC TGGTAGATGT GGGCGGTGTT TGGAGGCTGT    #            1750TGCATAT ACTGTGAGCT GTGATTGGGG AACACCAATG    #            1800CAGGCAG CTAAGCAGCA CCTCAAGAAA ACATGTTAAA    #            1850TCTTACA GTAGTTCAAA TACAAAACTG AAATGAAATC    #                 187 - #0    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 477 amino              (B) TYPE: Amino Acid              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    #Ala Met Ala Arg Leule Arg Trp His Gly Pro    #15    #Trp Arg Ala Ala Pherp Leu Val Val Gly Phe    #30    #Ser Arg Ile Trp Cyser Cys Lys Cys Ser Ala    #45    #Pro Arg Leu Glu Proro Gly Ile Val Ala Phe    #60    #Ile Phe Ile Ala Asnro Glu Asn Ile Thr Glu    #75    #Asp Val Glu Ala Tyrlu Ile Ile Asn Glu Asp    #90    #Ser Gly Leu Lys Phesn Leu Thr Ile Val Asp    #95                 1 - #00                 1 - #05    #Asn Leu Gln His Ilela Phe Leu Lys Asn Ser    #                120    #Ser Arg Lys His Phesn Lys Leu Thr Ser Leu    #                135    #Val Gly Asn Pro Pheeu Ser Glu Leu Ile Leu    #                150    #Thr Leu Gln Glu Alasp Ile Met Trp Ile Lys    #                165    #Cys Leu Asn Glu Sersp Thr Gln Asp Leu Tyr    #                180    #Ile Pro Asn Cys Glyro Leu Ala Asn Leu Gln    #                195    #Leu Thr Val Glu Glusn Leu Ala Ala Pro Asn    #                210    #Ala Gly Asp Pro Valhr Leu Ser Cys Ser Val    #                225    #Val Ser Lys His Metrp Asp Val Gly Asn Leu    #                240    #Arg Ile Thr Asn Ileis Thr Gln Gly Ser Leu    #                255    #Cys Val Ala Glu Asner Gly Lys Gln Ile Ser    #                270    #Leu Thr Val His Phesp Gln Asp Ser Val Asn    #                285    #Thr Ser Asp His Hishr Phe Leu Glu Ser Pro    #                300    #Pro Lys Pro Ala Leuhe Thr Val Lys Gly Asn    #                315    #Glu Ser Lys Tyr Ilesn Gly Ala Ile Leu Asn    #                330    #Glu Tyr His Gly Cysis Val Thr Asn His Thr    #                345    #Asn Gly Asp Tyr Thrsn Pro Thr His Met Asn    #                360    #Glu Lys Gln Ile Sersn Glu Tyr Gly Lys Asp    #                375    #Asp Gly Ala Asn Proly Trp Pro Gly Ile Asp    #                390    #Gly Thr Ala Ala Asnal Ile Tyr Glu Asp Tyr    #                405    #Glu Ile Pro Ser Thrhr Thr Asn Arg Ser Asn    #                420    #Leu Ser Val Tyr Alays Thr Gly Arg Glu His    #                435    #Cys Leu Leu Val Metla Ser Val Val Gly Phe    #                450    #Lys Phe Gly Met Lysys Leu Ala Arg His Ser    #                465    #Asp Glyhe Val Leu Phe His Lys Ile Pro Leu    #                475    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 2715 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    #              50AGATGGA TGTCTCTCTT TGCCCAGCCA AGTGTAGTTT    #             100TTGCTGG GAAGCGTCTG GCTGGACTAT GTGGGCTCCG    #             150TGCAAAT TGTGTCTGCA GCAAGACTGA GATCAATTGC    #             200ATGGGAA CCTCTTCCCC CTCCTGGAAG GGCAGGATTC    #             250GGGAACG CCAATATCAA CATCACGGAC ATCTCAAGGA    #             300ACACATA GAGAACTGGC GCAGTCTTCA CACGCTCAAC    #             350AGCTCTA CACCGGACTT CAAAAGCTGA CCATCAAGAA    #             400AGCATTC AGCCCAGAGC CTTTGCCAAG AACCCCCATT    #             450CCTGTCA AGTAACCGGC TCACCACACT CTCGTGGCAG    #             500TGAGTCT TCGGGAATTG CAGTTGGAGC AGAACTTTTT    #             550GACATCC GCTGGATGCA GCTCTGGCAG GAGCAGGGGG    #             600CAGCCAG AACCTCTACT GCATCAATGC TGATGGCTCC    #             650TCCGCAT GAACATCAGT CAGTGTGACC TTCCTGAGAT    #             700GTCAACC TGACCGTACG AGAGGGTGAC AATGCTGTTA    #             750CTCTGGA TCACCCCTTC CTGATGTGGA CTGGATAGTC    #             800CCATCAA CACTCACCAG ACCAATCTGA ACTGGACCAA    #             850AACTTGA CGCTGGTGAA TGTGACGAGT GAGGACAATG    #             900GTGCATT GCAGAGAACG TGGTGGGCAT GAGCAATGCC    #             950CTGTCTA CTATCCCCCA CGTGTGGTGA GCCTGGAGGA    #            1000CTGGAGC ACTGCATCGA GTTTGTGGTG CGTGGCAACC    #            1050GCACTGG CTGCACAATG GGCAGCCTCT GCGGGAGTCC    #            1100TGGAATA CTACCAAGAG GGAGAGATTT CCGAGGGCTG    #            1150AAGCCCA CCCACTACAA CAATGGCAAC TATACCCTCA    #            1200ACTGGGC ACAGCCAACC AGACCATCAA TGGCCACTTC    #            1250TTCCAGA GAGCACGGAT AACTTTATCT TGTTTGACGA    #            1300CCTCCTA TCACTGTGAC CCACAAACCA GAAGAAGACA    #            1350CATAGCA GTTGGACTTG CTGCTTTTGC CTGTGTCCTG    #            1400TCGTCAT GATCAACAAA TATGGTCGAC GGTCCAAATT    #            1450CCCGTGG CTGTCATCAG TGGTGAGGAG GACTCAGCCA    #            1500CATCAAC CACGGCATCA CCACGCCCTC GTCACTGGAT    #            1550CTGTGGT CATTGGCATG ACTCGCATCC CTGTCATTGA    #            1600TTCCGTC AGGGACACAA CTGCCACAAG CCGGACACGT    #            1650TAAGAGG AGAGACATCG TGCTGAAGCG AGAACTGGGT    #            1700GAAAGGT CTTCCTGGCC GAGTGCTACA ACCTCAGCCC    #            1750ATGCTTG TGGCTGTGAA GGCCCTGAAG GATCCCACCC    #            1800GGATTTC CAGAGGGAGG CCGAGCTGCT CACCAACCTG    #            1850TTGTCAA GTTCTATGGA GTGTGCGGCG ATGGGGACCC    #            1900TTTGAAT ACATGAAGCA TGGAGACCTG AATAAGTTCC    #            1950GCCAGAT GCAATGATCC TTGTGGATGG ACAGCCACGC    #            2000AGCTGGG GCTCTCCCAA ATGCTCCACA TTGCCAGTCA    #            2050ATGGTGT ACCTGGCCTC CCAGCACTTT GTGCACCGAG    #            2100GAACTGC CTGGTTGGAG CGAATCTGCT AGTGAAGATT    #            2150TGTCCAG AGATGTCTAC AGCACGGATT ATTACAGGCT    #            2200GGAAATG ATTTTTGTAT ATGGTGTGAG GTGGGAGGAC    #            2250CATTCGC TGGATGCCTC CTGAAAGCAT CATGTACCGG    #            2300AGAGTGA TGTATGGAGC TTCGGGGTGA TCCTCTGGGA    #            2350GGAAAGC AGCCATGGTT CCAACTCTCA AACACGGAGG    #            2400TACCCAA GGTCGTGTTT TGGAGCGGCC CCGAGTCTGC    #            2450ACGATGT CATGCTGGGG TGCTGGCAGA GGGAACCACA    #            2500ATCAAGG AGATCTACAA AATCCTCCAT GCTTTGGGGA    #            2550CTACCTG GACATTCTTG GCTAGTGGTG GCTGGTGGTC    #            2600CTGTTGC CTCCTCTCTC CCTGCCTCAC ATCTCCCTTC    #            2650CTTCCAT CCTTGACTGA AGCGAACATC TTCATATAAA    #            2700TACACAT ACAACACTGA AAAAAGGAAA AAAAAAGAAA    #  2715    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 839 amino              (B) TYPE: Amino Acid              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    #Ser Phe Trp Arg Ileeu Cys Pro Ala Lys Cys    #15    #Val Gly Ser Val Leuer Val Trp Leu Asp Tyr    #30    #Thr Glu Ile Asn Cyssn Cys Val Cys Ser Lys    #45    #Leu Leu Glu Gly Glnsp Gly Asn Leu Phe Pro    #60    #Ile Asn Ile Thr Asper Asn Gly Asn Ala Asn    #75    #Glu Asn Trp Arg Serle Thr Ser Ile His Ile    #90    #Leu Tyr Thr Gly Leusn Ala Val Asp Met Glu    #95                 1 - #00                 1 - #05    #Arg Ser Ile Gln Prole Lys Asn Ser Gly Leu    #                120    #Tyr Ile Asn Leu Serys Asn Pro His Leu Arg    #                135    #Leu Phe Gln Thr Leuhr Thr Leu Ser Trp Gln    #                150    #Phe Phe Asn Cys Sereu Gln Leu Glu Gln Asn    #                165    #Glu Gln Gly Glu Alarp Met Gln Leu Trp Gln    #                180    #Asn Ala Asp Gly Serln Asn Leu Tyr Cys Ile    #                195    #Gln Cys Asp Leu Prohe Arg Met Asn Ile Ser    #                210    #Val Arg Glu Gly Asper His Val Asn Leu Thr    #                225    #Ser Pro Leu Pro Asphr Cys Asn Gly Ser Gly    #                240    #Ile Asn Thr His Glnal Thr Gly Leu Gln Ser    #                255    #Ile Asn Leu Thr Leurp Thr Asn Val His Ala    #                270    #Thr Leu Thr Cys Ileer Glu Asp Asn Gly Phe    #                285    #Ser Val Ala Leu Thral Gly Met Ser Asn Ala    #                300    #Glu Glu Pro Glu Leuro Arg Val Val Ser Leu    #                315    #Arg Gly Asn Pro Proys Ile Glu Phe Val Val    #                330    #Pro Leu Arg Glu Serrp Leu His Asn Gly Gln    #                345    #Gly Glu Ile Ser Glual Glu Tyr Tyr Gln Glu    #                360    #Tyr Asn Asn Gly Asnhe Asn Lys Pro Thr His    #                375    #Thr Ala Asn Gln Thrla Lys Asn Pro Leu Gly    #                390    #Pro Glu Ser Thr Asphe Leu Lys Glu Pro Phe    #                405    #Thr Pro Pro Ile Thrhe Asp Glu Val Ser Pro    #                420    #Gly Val Ser Ile Alaro Glu Glu Asp Thr Phe    #                435    #Leu Val Val Leu Phela Phe Ala Cys Val Leu    #                450    #Lys Phe Gly Met Lysys Tyr Gly Arg Arg Ser    #                465    #Asp Ser Ala Ser Proal Ile Ser Gly Glu Glu    #                480    #Pro Ser Ser Leu Aspsn His Gly Ile Thr Thr    #                495    #Thr Arg Ile Pro Valhr Val Val Ile Gly Met    #                510    #His Asn Cys His Lysln Tyr Phe Arg Gln Gly    #                525    #Arg Asp Ile Val Leual Gln His Ile Lys Arg    #                540    #Lys Val Phe Leu Alaly Glu Gly Ala Phe Gly    #                555    #Lys Met Leu Val Alaeu Ser Pro Thr Lys Asp    #                570    #Ala Arg Lys Asp Pheys Asp Pro Thr Leu Ala    #                585    #Gln His Glu His Ilelu Leu Leu Thr Asn Leu    #                600    #Asp Pro Leu Ile Metly Val Cys Gly Asp Gly    #                615    #Asn Lys Phe Leu Arget Lys His Gly Asp Leu    #                630    #Asp Gly Gln Pro Argsp Ala Met Ile Leu Val    #                645    #Met Leu His Ile Alalu Leu Gly Leu Ser Gln    #                660    #Ala Ser Gln His Pheer Gly Met Val Tyr Leu    #                675    #Leu Val Gly Ala Asneu Ala Thr Arg Asn Cys    #                690    #Ser Arg Asp Val Tyrle Gly Asp Phe Gly Met    #                705    #Ser Gly Asn Asp Pheyr Arg Leu Phe Asn Pro    #                720    #Met Leu Pro Ile Arglu Val Gly Gly His Thr    #                735    #Lys Phe Thr Thr Glulu Ser Ile Met Tyr Arg    #                750    #Trp Glu Ile Phe Threr Phe Gly Val Ile Leu    #                765    #Asn Thr Glu Val Ilero Trp Phe Gln Leu Ser    #                780    #Arg Pro Arg Val Cysln Gly Arg Val Leu Glu    #                795    #Cys Trp Gln Arg Gluyr Asp Val Met Leu Gly    #                810    #Tyr Lys Ile Leu Hiseu Asn Ile Lys Glu Ile    #                825    #Asp Ile Leu Glyys Ala Thr Pro Ile Tyr Leu    #                835    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1858 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    #              50AGATGGA TGTCTCTCTT TGCCCAGCCA AGTGTAGTTT    #             100TTGCTGG GAAGCGTCTG GCTGGACTAT GTGGGCTCCG    #             150TGCAAAT TGTGTCTGCA GCAAGACTGA GATCAATTGC    #             200ATGGGAA CCTCTTCCCC CTCCTGGAAG GGCAGGATTC    #             250GGGAACG CCAATATCAA CATCACGGAC ATCTCAAGGA    #             300ACACATA GAGAACTGGC GCAGTCTTCA CACGCTCAAC    #             350AGCTCTA CACCGGACTT CAAAAGCTGA CCATCAAGAA    #             400AGCATTC AGCCCAGAGC CTTTGCCAAG AACCCCCATT    #             450CCTGTCA AGTAACCGGC TCACCACACT CTCGTGGCAG    #             500TGAGTCT TCGGGAATTG CAGTTGGAGC AGAACTTTTT    #             550GACATCC GCTGGATGCA GCTCTGGCAG GAGCAGGGGG    #             600CAGCCAG AACCTCTACT GCATCAATGC TGATGGCTCC    #             650TCCGCAT GAACATCAGT CAGTGTGACC TTCCTGAGAT    #             700GTCAACC TGACCGTACG AGAGGGTGAC AATGCTGTTA    #             750CTCTGGA TCACCCCTTC CTGATGTGGA CTGGATAGTC    #             800CCATCAA CACTCACCAG ACCAATCTGA ACTGGACCAA    #             850AACTTGA CGCTGGTGAA TGTGACGAGT GAGGACAATG    #             900GTGCATT GCAGAGAACG TGGTGGGCAT GAGCAATGCC    #             950CTGTCTA CTATCCCCCA CGTGTGGTGA GCCTGGAGGA    #            1000CTGGAGC ACTGCATCGA GTTTGTGGTG CGTGGCAACC    #            1050GCACTGG CTGCACAATG GGCAGCCTCT GCGGGAGTCC    #            1100TGGAATA CTACCAAGAG GGAGAGATTT CCGAGGGCTG    #            1150AAGCCCA CCCACTACAA CAATGGCAAC TATACCCTCA    #            1200ACTGGGC ACAGCCAACC AGACCATCAA TGGCCACTTC    #            1250TTCCAGA GAGCACGGAT AACTTTATCT TGTTTGACGA    #            1300CCTCCTA TCACTGTGAC CCACAAACCA GAAGAAGACA    #            1350CATAGCA GTTGGACTTG CTGCTTTTGC CTGTGTCCTG    #            1400TCGTCAT GATCAACAAA TATGGTCGAC GGTCCAAATT    #            1450CCCGTGG CTGTCATCAG TGGTGAGGAG GACTCAGCCA    #            1500CATCAAC CACGGCATCA CCACGCCCTC GTCACTGGAT    #            1550CTGTGGT CATTGGCATG ACTCGCATCC CTGTCATTGA    #            1600TTCCGTC AGGGACACAA CTGCCACAAG CCGGACACGT    #            1650CATAGAC AATCATGGGA TATTAAACTT GAAGGACAAT    #            1700TCCCATC AACTCACTAT ATATATGAGG AACCTGAGGT    #            1750GTGTCTT ACCCAAGGTC ACATGGTTTC AGAGAAATTA    #            1800AAGCCTT CCCGGACATT CCAAGCCTCT TAACCATGGC    #            1850ATGTCAA TGTTTATTTC AGCAAAGGAC GTCATGGCCT    #        1858    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 612 amino              (B) TYPE: Amino Acid              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    #Ser Phe Trp Arg Ileeu Cys Pro Ala Lys Cys    #15    #Val Gly Ser Val Leuer Val Trp Leu Asp Tyr    #30    #Thr Glu Ile Asn Cyssn Cys Val Cys Ser Lys    #45    #Leu Leu Glu Gly Glnsp Gly Asn Leu Phe Pro    #60    #Ile Asn Ile Thr Asper Asn Gly Asn Ala Asn    #75    #Glu Asn Trp Arg Serle Thr Ser Ile His Ile    #90    #Leu Tyr Thr Gly Leusn Ala Val Asp Met Glu    #95                 1 - #00                 1 - #05    #Arg Ser Ile Gln Prole Lys Asn Ser Gly Leu    #                120    #Tyr Ile Asn Leu Serys Asn Pro His Leu Arg    #                135    #Leu Phe Gln Thr Leuhr Thr Leu Ser Trp Gln    #                150    #Phe Phe Asn Cys Sereu Gln Leu Glu Gln Asn    #                165    #Glu Gln Gly Glu Alarp Met Gln Leu Trp Gln    #                180    #Asn Ala Asp Gly Serln Asn Leu Tyr Cys Ile    #                195    #Gln Cys Asp Leu Prohe Arg Met Asn Ile Ser    #                210    #Val Arg Glu Gly Asper His Val Asn Leu Thr    #                225    #Ser Pro Leu Pro Asphr Cys Asn Gly Ser Gly    #                240    #Ile Asn Thr His Glnal Thr Gly Leu Gln Ser    #                255    #Ile Asn Leu Thr Leurp Thr Asn Val His Ala    #                270    #Thr Leu Thr Cys Ileer Glu Asp Asn Gly Phe    #                285    #Ser Val Ala Leu Thral Gly Met Ser Asn Ala    #                300    #Glu Glu Pro Glu Leuro Arg Val Val Ser Leu    #                315    #Arg Gly Asn Pro Proys Ile Glu Phe Val Val    #                330    #Pro Leu Arg Glu Serrp Leu His Asn Gly Gln    #                345    #Gly Glu Ile Ser Glual Glu Tyr Tyr Gln Glu    #                360    #Tyr Asn Asn Gly Asnhe Asn Lys Pro Thr His    #                375    #Thr Ala Asn Gln Thrla Lys Asn Pro Leu Gly    #                390    #Pro Glu Ser Thr Asphe Leu Lys Glu Pro Phe    #                405    #Thr Pro Pro Ile Thrhe Asp Glu Val Ser Pro    #                420    #Gly Val Ser Ile Alaro Glu Glu Asp Thr Phe    #                435    #Leu Val Val Leu Phela Phe Ala Cys Val Leu    #                450    #Lys Phe Gly Met Lysys Tyr Gly Arg Arg Ser    #                465    #Asp Ser Ala Ser Proal Ile Ser Gly Glu Glu    #                480    #Pro Ser Ser Leu Aspsn His Gly Ile Thr Thr    #                495    #Thr Arg Ile Pro Valhr Val Val Ile Gly Met    #                510    #His Asn Cys His Lysln Tyr Phe Arg Gln Gly    #                525    #Asn His Gly Ile Leual Phe Ser Asn Ile Asp    #                540    #Pro Ser Thr His Tyrsn Arg Asp His Leu Val    #                555    #Glu Val Ser Tyr Proro Glu Val Gln Ser Gly    #                570    #Asn Pro Ile Ser Leuhe Arg Glu Ile Met Leu    #                585    #Ile Tyr Val Glu Aspys Pro Leu Asn His Gly    #                600    #Gly Phesn Val Tyr Phe Ser Lys Gly Arg His    #                610    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 790 amino              (B) TYPE: Amino Acid              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    #Gly Trp His Ser Trply Arg Arg Gly Gln Leu    #15    #Leu Ile Leu Ala Serly Ser Leu Leu Ala Trp    #30    #Cys Pro His Gly Serro Cys Pro Asp Ala Cys    #45    #Leu Asp Ser Leu Hisys Thr Arg Asp Gly Ala    #60    #Leu Tyr Ile Glu Asnla Glu Asn Leu Thr Glu    #75    #Asp Leu Arg Gly Leuln His Leu Glu Leu Arg    #90    #Ser Gly Leu Arg Phesn Leu Thr Ile Val Lys    #95                 1 - #00                 1 - #05    #Arg Leu Ser Arg Leula Phe His Phe Thr Pro    #                120    #Ser Trp Lys Thr Valsn Ala Leu Glu Ser Leu    #                135    #Ser Gly Asn Pro Leueu Gln Glu Leu Val Leu    #                150    #Arg Trp Glu Glu Glula Leu Arg Trp Leu Gln    #                165    #Gln Cys His Gly Glnal Pro Glu Gln Lys Leu    #                180    #Cys Gly Val Pro Thris Met Pro Asn Ala Ser    #                195    #Asp Val Gly Asp Aspal Pro Asn Ala Ser Val    #                210    #Gly Leu Glu Gln Alays Gln Val Glu Gly Arg    #                225    #Ala Thr Val Met Lyshr Glu Leu Glu Gln Ser    #                240    #Leu Ala Asn Val Thrro Ser Leu Gly Leu Thr    #                255    #Trp Ala Glu Asn Asprg Lys Asn Leu Thr Cys    #                270    #Asn Val Ser Phe Prolu Val Ser Val Gln Val    #                285    #Met His His Trp Cyseu His Thr Ala Val Glu    #                300    #Pro Ser Leu Arg Trpal Asp Gly Gln Pro Ala    #                315    #Ser Phe Ile Phe Threr Val Leu Asn Glu Thr    #                330    #Val Arg His Gly Cysro Ala Ala Asn Glu Thr    #                345    #Asn Gly Asn Tyr Thrln Pro Thr His Val Asn    #                360    #Ser Ala Ser Ile Metsn Pro Phe Gly Gln Ala    #                375    #Asn Pro Glu Asp Prosp Asn Pro Phe Glu Phe    #                390    #Pro Val Glu Lys Lyssn Ser Thr Ser Gly Asp    #                405    #Val Gly Leu Ala Valhe Gly Val Ser Val Ala    #                420    #Leu Val Leu Asn Lyshe Leu Ser Thr Leu Leu    #                435    #Arg Pro Ala Val Leusn Lys Phe Gly Ile Asn    #                450    #His Phe Met Thr Leuly Leu Ala Met Ser Leu    #                465    #Lys Gly Ser Gly Leueu Ser Pro Thr Glu Gly    #                480    #Phe Ser Asp Ala Cysle Glu Asn Pro Gln Tyr    #                495    #Leu Lys Trp Glu Leuys Arg Arg Asp Ile Val    #                510    #Ala Glu Cys His Asnhe Gly Lys Val Phe Leu    #                525    #Ala Val Lys Ala Leuln Asp Lys Met Leu Val    #                540    #Phe Gln Arg Glu Alalu Ser Ala Arg Gln Asp    #                555    #Ile Val Arg Phe Pheet Leu Gln His Gln His    #                570    #Met Val Phe Glu Tyrlu Gly Arg Pro Leu Leu    #                585    #Arg Ser His Gly Prosp Leu Asn Arg Phe Leu    #                600    #Val Ala Pro Gly Proeu Ala Gly Gly Glu Asp    #                615    #Ser Gln Val Ala Alaln Leu Leu Ala Val Ala    #                630    #Val His Arg Asp Leueu Ala Gly Leu His Phe    #                645    #Leu Val Val Lys Ileys Leu Val Gly Gln Gly    #                660    #Ser Thr Asp Tyr Tyret Ser Arg Asp Ile Tyr    #                675    #Arg Trp Met Pro Prorg Thr Met Leu Pro Ile    #                690    #Glu Ser Asp Val Trpyr Arg Lys Phe Thr Thr    #                705    #Thr Tyr Gly Lys Glnal Leu Trp Glu Ile Phe    #                720    #Ile Asp Cys Ile Threu Ser Asn Thr Glu Ala    #                735    #Cys Pro Pro Glu Valeu Glu Arg Pro Arg Ala    #                750    #Glu Pro Gln Gln Argrg Gly Cys Trp Gln Arg    #                765    #Gln Ala Leu Ala Glnsp Val His Ala Arg Leu    #                780    -  Ala Pro Pro Val Tyr Leu Asp Val Leu Gly    #                790    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    #                23YTNAA RAC    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    -  TGGATGCARY TNTGGCARCA RCA 23    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    #21                YTCRT T    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    #                23TCNAC GTG    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    #                 22ACTA CA    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 25 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    #               25 AGATT TCTGC    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    #                23CCTCA AGG    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    #                 22TTCT CC    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    #                24GGTTG TATC    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    #                 22GTGG GA    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    # 19               CCCC    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    # 19               GGAA    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    # 19               TGAG    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    # 20               CCTGG    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    # 20               GGCAG    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    #                 22CCAC CA    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    # 20               GATGC    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    #                24AGGTG GCAT    - (2) INFORMATION FOR SEQ ID NO:28:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    #21                AACCT C    - (2) INFORMATION FOR SEQ ID NO:29:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    #21                CCAGC C    - (2) INFORMATION FOR SEQ ID NO:30:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    #21                AGTGA C    - (2) INFORMATION FOR SEQ ID NO:31:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    #                23CGCAG AGG    - (2) INFORMATION FOR SEQ ID NO:32:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    #21                TCCGG T    - (2) INFORMATION FOR SEQ ID NO:33:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    #21                AGAGG A    - (2) INFORMATION FOR SEQ ID NO:34:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 24 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    #                24AGCCA TCCA    - (2) INFORMATION FOR SEQ ID NO:35:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    # 19               AATG    - (2) INFORMATION FOR SEQ ID NO:36:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 9 amino              (B) TYPE: Amino Acid              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    -  Glu Ser Thr Asp Asn Phe Ile Leu Phe    #5 1    - (2) INFORMATION FOR SEQ ID NO:37:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 14 amino              (B) TYPE: Amino Acid              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    #Ile Trp Cys Gluro Ser Gly Asn Asp Phe Cys    #10    - (2) INFORMATION FOR SEQ ID NO:38:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: Nucleic Acid              (C) STRANDEDNESS: Single              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    #  18              TGG    - (2) INFORMATION FOR SEQ ID NO:39:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 6 amino              (B) TYPE: Amino Acid              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    -  Ser Pro Ser Arg Arg Trp    #5 1    - (2) INFORMATION FOR SEQ ID NO:40:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 11 amino              (B) TYPE: Amino Acid              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    #Glyhe Val Leu Phe His Lys Ile Pro Leu Asp    #10    - (2) INFORMATION FOR SEQ ID NO:41:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 84 amino              (B) TYPE: Amino Acid              (D) TOPOLOGY: Linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    #Ile Leu Asn Leu Lyssn Ile Asp Asn His Gly    #15    #His Tyr Ile Tyr Gluis Leu Val Pro Ser Thr    #30    #Tyr Pro Arg Ser Hisln Ser Gly Glu Val Ser    #45    #Ser Leu Pro Gly Hisle Met Leu Asn Pro Ile    #60    #Glu Asp Val Asn Valsn His Gly Ile Tyr Val    #75    -  Tyr Phe Ser Lys Gly Arg His Gly Phe    #80    __________________________________________________________________________

We claim:
 1. A monoclonal antibody or antigen-binding fragment thereof,that specifically binds a polypeptide comprising an amino acid sequenceselected from the group consisting of:(a) mature human trkB in SEQ IDNO: 2; (b) mature human trkC in SEQ ID NO: 6; (c) trk B or trk Cpolyphide encode by a naturally-occurring allelic variant or splicevariant of (a) or (b); and, (d) a neurotrophin-binding, secondimmunoglobulin-like C2-type domain of (a), (b), or (c).
 2. A hybridomacell line producing the antibody or antigen-binding fragment of claim 1.3. A composition comprising an antibody or antigen-binding fragment ofclaim 1 in admixture with a physiologically acceptable carrier.
 4. Theantibody or antigen-binding fragment of claim 1 that is an antagonist ofhuman trkB or human trkC.
 5. The antibody or antigen-binding fragment ofclaim 1 that is humanized.
 6. The antibody or antigen-binding fragmentof claim 1 that is a human antibody or antigen-binding fragment thereof.7. The antibody or antigen-binding fragment of claim 1, wherein theselected amino acid sequence is (a) or (b).
 8. The antibody orantigen-binding fragment of claim 7, wherein the selected amino acidsequence is (b).
 9. The antibody or antigen-binding fragment of claim 1,wherein the selected amino acid sequence is from (c).
 10. The antibodyor antigen-binding fragment of claim 9, wherein the selected amino acidsequence from (c) is encoded by a naturally-occurring allelic variant orsplice variant of (b).
 11. The monoclonal antibody or antigen-bindingfragment of claim 1, wherein the amino acid sequence is selected thegroup consisting of:(a) mature human trkC in SEQ ID NO: 6; (b) trkCpolypeptide encoded by a naturally-occurring allelic variant or splicevariant of (a); and (c) a neurotrophin-binding, secondimmunoglobulin-like C2-type domain of (a) or (b).
 12. The monoclonalantibody or antigen-binding fragment of claim 11, wherein the amino acidis selected from group (c).
 13. A monoclonal antibody or antigen-bindingfragment thereof that specifically binds to a polypeptide having anamino acid sequence selected from the group consisting of:(a) from aboutamino acid residue 301 to about amino acid residue 365 in SEQ. ID. NO:2; and (b) from about amino acid residue 313 to about amino acid residue376 in SEQ. ID. NO:
 6. 14. A monoclonal antibody or antigen-bindingfragment thereof that specifically binds a polypeptide comprising anamino acid sequence selected from the group consisting of:(a) maturehuman trkB in SEQ ID NO: 2; (b) mature human trkC in SEQ ID NO: 6; (c)trkB or trkC polypephide encoded by a naturally-occurring allelicvariant or splice variant of (a) or (b); and (d) a neurotrophin-binding,second immunoglobulin-like C2-type domain of (a), (b), or (c),whereinthe antibody or antigen-binding fragment thereof is an agonist of humantrkC or human trkB.
 15. The monoclonal antibody of claim 14, wherein theantibody or antigen-binding fragment is an agonist of human trkC.